Proteomic Screening for Redox State Dependent Protein-Protein Interactions

ABSTRACT

This invention provides a modified yeast two-hybrid system in order to identify NO-dependent protein-protein interactions. Bait proteins implicated in apoptotic signaling pathways were used to identify NO-dependent interactions. The physiological relevance of these interactions is demonstrated by their occurrence and dependence on endogenous NO in mammalian cells, and by the functional interrelatedness of bait and prey.

RELATED APPLICATIONS

This application claims the benefit of PCT/US02/31571 filed Oct. 15,2002. The contents of this application is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention is directed to methods of identifying proteins involvedin, or representing markers for, disease, drug reactions, neoplasm(tumor) or infection. This invention also relates to methods ofidentifying protein interactions and previously unknown receptors andligands.

BACKGROUND OF THE INVENTION

Signal transduction is often coordinated by multi-protein complexes(Kumar and Snyder, 2002; Gavin et al., 2002; Ho et al., 2002).Constitutive interactions among proteins function in the initiation ofsignaling, while inducible or regulated protein-protein interactions aregenerally required for signal processing and propagation (Hunter, 2000).These dynamic interactions form the basis of complex regulatory circuitsthat determine biological function. Identifying the makeup of thesecircuits is a major challenge, however, because they involve multiplelow affinity interactions that are controlled by dynamicpost-translational protein modifications in response to multiple stimuli(Hunter, 2000; Zhu et al., 2001).

A number of non-covalent bonds form between proteins when two proteinsurfaces are precisely matched, and these bonds account for thespecificity of recognition. Protein-protein interactions are involvedin, for example, the assembly of enzyme subunits, antigen-antibodyreactions, forming the supramolecular structures of ribosomes,filaments, and viruses in transport, and in the interaction of receptorson a cell with growth factors and hormones. Products of oncogenes cangive rise to neoplastic transformation through protein-proteininteractions. For example, some oncogenes encode protein kinases whoseenzymatic activity on cellular target proteins leads to the cancerousstate. Another example of a protein-protein interaction occurs when avirus infects a cell by recognizing a polypeptide receptor on thesurface, and this interaction has been used to design antiviral agents.

Protein-protein interactions have been generally studied in the pastusing biochemical techniques such as cross-linking,co-immunoprecipitation and co-fractionation by chromatography. Adisadvantage of these techniques is that interacting proteins oftenexist in very low abundance and are, therefore, difficult to detect.Another major disadvantage is that these biochemical techniques involveonly the proteins, not the genes encoding them. When an interaction isdetected using biochemical methods, the newly identified protein oftenmust be painstakingly isolated and then sequenced to enable the geneencoding it to be obtained. Another disadvantage is that these methodsdo not immediately provide information about which domains of theinteracting proteins are involved in the interaction. Also, smallchanges in the composition of the interacting proteins cannot be testedeasily for their effect on the interaction.

A genetic system that is capable of rapidly detecting which proteinsinteract with a known protein, determining which domains of the proteinsinteract under physiological conditions, and providing the genes for thenewly identified interacting proteins has only recently been madeavailable. The yeast two-hybrid system currently represents the mostpowerful in vivo approach to screen for polypeptides that could bind toa given target protein and this invention provides a unique way ofutilizing the two hybrid system for studying novel protein-proteininteractions under physiological conditions.

The yeast two hybrid system described here is based on transcriptionalactivation. Transcription is the process by which RNA molecules aresynthesized using a DNA template. Transcription is regulated by specificsequences in the DNA which indicate when and where RNA synthesis shouldbegin. These sequences correspond to binding sites for proteins,designated transcription factors, which interact with the enzymaticmachinery used for the RNA polymerization reaction.

In these systems, reconstitution into a hybrid caused by protein-proteininteraction of a bait protein with a prey protein is monitored byactivation of a reporter gene. Two-hybrid systems are discussed, forexample, in Nandabalan et al U.S. Pat. No. 6,083,693; U.S. Pat. No.5,283,173; U.S. Pat. No. 5,610,015; U.S. Pat. No. 5,634,463; U.S. Pat.No. 5,885,779; Klein et al. United States Statutory InventionRegistration H1,892; LeGrain et al U.S. Pat. No. 6,187,535; and Rain,J.-C., et al. Nature 409, 211-215 (Jan. 11, 2001). The bait is a proteinor proteins known to be involved in the pathophysiological process forwhich the determination is being made. The prey can be constituted ofall proteins and genes expressed in cells of an affected tissue or bodyfluid or an election therefrom. Other methods of determiningprotein-protein interactions (e.g., as described in Zhu, H, et al.,Science 293, 210 1-2105 (2001) and as described below) can also be used.In general, the bait protein is derived from genomic DNA or a cDNAlibrary. The cDNA library can be derived from a cell, for example, amacrophage, a cytokine activated macrophage, an endothelial cell, amuscle cell, a tumor cell or a kidney cell. Alternatively, the cDNAlibrary can be derived from a cell treated with a drug, preferably achemotherapeutic drug such as cisplatin.

In essence, the two putative protein partners are genetically fused tothe DNA-binding domain of a transcription factor and to atranscriptional activation domain, respectively. A productiveinteraction between the two proteins of interest will bring thetranscriptional activation domain into the proximity of the DNA-bindingdomain and will trigger directly the transcription of an adjacentreporter gene, for example, lacZ, giving a screenable phenotype. Thetranscription can be activated through the use of two functional domainsof a transcription factor: a domain that recognizes and binds to aspecific site on the DNA and a domain that is necessary for activation,as reported by Keegan et al. (1986) and Ma et al. (1987).

Transcriptional activation has been studied using the GAL4 protein ofthe yeast Saccharomyces cerevisiae (S. cerevisiae). The GAL4 protein isa transcriptional activator required for the expression of genesencoding enzymes of galactose utilization, see Johnston, Microbiol.Rev., 51, 458-476 (1987). It consists of an N-terminal domain whichbinds to specific DNA sequences designated UAS_(G), (UAS stands forupstream activation site, G indicates the galactose genes) and aC-terminal domain containing acidic regions, which is necessary toactivate transcription, see Keegan et al. (1986), supra., and Ma andPtashne. (1987), supra. As discussed by Keegan et al., the N-terminaldomain binds to DNA in a sequence-specific manner but fails to activatetranscription. The C-terminal domain cannot activate transcriptionbecause it fails to localize to the UAS_(G), see for example, Brent andPtashne, Cell, 43, 729-736 (1985). However, Ma and Ptashne have reported(Cell, 51, 113-119 (1987); Cell, 55, 443-446 (1988)) that when both theGAL4 N-terminal domain and C-terminal domain are fused together in thesame protein, transcriptional activity is induced. Other proteins alsofunction as transcriptional activators via the same mechanism. Forexample, the GCN4 protein of Saccharomyces cerevisiae as reported byHope and Struhl, Cell, 46, 885-894 (1986), the ADR1 protein ofSaccharomyces cerevisiae as reported by Thukral et al., Molecular andCellular Biology, 9, 2360-2369, (1989) and the human estrogen receptor,as discussed by Kumar et al., Cell, 51, 941-951 (1987) both containseparable domains for DNA binding and for maximal transcriptionalactivation.

Recently, Rossi et al. (1997) described a different approach, amammalian “two-hybrid” system, which uses β-galactosidasecomplementation (Ullmann et al., 1968) to monitor protein-proteininteractions in intact eukaryotic cells. The number of genome sequencesof prokaryotic as well as eukaryotic host organisms available isincreasing exponentially and there is a great need for new toolsdirected to the functional and global study of these newly characterizedcomplete or partial genomes.

Systems for determining protein-protein interactions which are usefulherein are also described in Fung, E. T., et al, Current Opinion inBiotechnology 12:65-69 (2001) and Delneri, I., et al, Current Opinion inBiotechnology 12:87-9 1 (2001). Systems for determining proteininteractions or activity include those described in Sakura, T., et al.,Cell (1998), 573-5 85 and Hare, 1, et al., Nature Medicine, Vol 5,1241-1242 (1999). These systems involve a search for an orphan receptoror ligand where readout is measured by changes in an intracellularsecond messenger such as calcium or G-protein activity. Other systemsfor determining protein interactions or activity include those describedin Scherer, P., et al, Nature Biotechnology 16, 58 1-586 (1998). Thesesystems involve a search for new epitopes that has been unmasked throughprotein-protein interaction.

Systems for determining changes in the level of protein expression aredescribed in Fung, E. T., et al, Current Opinion in Biotechnology 12:65-69 (2001). Systems for determining changes in the interaction betweenproteins and other molecules (e.g., DNA, RNA, lipids) are described inRen, B., et al, Science 290, 2306 (2000) and in Marshall, H. andStamler, J. S., Biochemistry 40, 1688 (2001). Methods for determininggenomic interactions include methods for assaying the expression ofgenes in differential display, e.g., as described in Zohinhofer, D., etal., Circulation, 103, 1396-1402 (2001) and SAGE where levels of mRNAare quantified through hybridization or other means of quantification,e.g., as described in Zhang, L., et al., Science Vol. 276, 1268-1272(1997).

Strategies for studying cellular function involved in disease statesgenerally rely on comparison of control and disease states. A number ofdifferent proteomic and genomic strategies, including differentialprofiling platforms and functional assays (e.g., interaction studies)have been routinely employed for this purpose. These assays have longrelied on the assumption that they accurately simulate thepathophysiological processes under investigation. However, they arecarried out in “open” air and, therefore, are performed in the absenceof specific and important protein modifications that are characteristicof physiological conditions, e.g., modifications that occur in thepresence of nitric oxide (NO).

Nitric oxide (NO) is a ubiquitous molecule that propagates its signalthrough post-translational nitrosylation of proteins (Stamler et al.,2001). Specifically, NO targets cysteine thiol and transition metalcenters to regulate a broad functional spectrum of substrates, includingall major classes of signaling proteins. An emerging theme in NO biologyis that NO synthases (NOS) are localized within multi-protein signalingcomplexes where they regulate signal transduction (Stamler et al.,2001). But whether NO can directly affect protein-protein interactionstransducing these signals, particularly in a disease state, has not beenpreviously considered.

Accordingly, current screening methodologies lack a level of validationand biological significance because the actions and interactionsidentified using prior art methods are not causally related to thepathophysiological processes. It is therefore an object of the presentinvention to describe methods for identifying protein interactions undermore physiological conditions. Specifically, this invention relates tothe utilization of, for example, a modified yeast two-hybrid screeningmethodology in order to assess the possibility of NO-dependentregulation of protein-protein interactions in a cellular context.

BRIEF SUMMARY OF THE INVENTION

This invention provides novel methods for identifying proteininteractions that are regulated by the redox state of the cell in whichthey occur and may, therefore, be regulated by a “redox state modifiermolecule” (RSMM). In general, modification of the redox state will beany change of the redox state compared to normal physiologicalconditions. Specifically, this invention provides a method foridentifying a protein complex whose formation is inhibited by an RSMM(“RSMM inhibited protein complex”); a method for identifying a proteincomplex whose formation is induced by an RSMM (“RSMM induced proteincomplex”); a method for identifying an agent capable of inhibiting RSMMinduction of a protein complex; and a method for identifying an agentcapable of inhibiting an RSMM inhibition of protein complex formation.

This invention provides a novel method for detecting an interactionbetween a first test protein and a second test protein in the presenceof an RSMM. RSMMs are those compounds produced in vivo that are oftencharacteristic of a pathophysiological process and which, because oftheir presence and/or concentration, affect a redox state. An RSMM isoften produced in enzymatic reactions associated with pathophysiologicalprocesses. Specific RSMMs may be implicated in some diseases but notothers. For example, certain oxidases are highly activated ininflammatory bowel disease, but not in atherosclerosis. A specificoxidase is responsible for bone resorption and another for hypertension.A certain diaphorase controls the activity of p53-dependent apoptoticdeath cascades (implicated in cancers) but is not implicated in otherapoptotic mechanisms which are p53-independent. Nitric oxide synthasecauses redox mediated damage in diabetes. Other enzymes involved indisease and other pathophysiological processes include oxygenases,peroxidases, reductases, transferases and dehydrogenases and the enzymesystems that control each of these kinds of enzymes.

This invention provides a genetically engineered and further modifiedtwo hybrid-based methodology to address the question of whether, forexample, NO can regulate protein-protein interactions. It also providesa method of screening for these interactions under physiologicallyrelevant conditions. This strategy has revealed multiple NO-dependentprotein-protein interactions, including interactions of nitric oxidesynthases (NOSs), whose physiological relevance is suggested by theiroccurrence and dependence on endogenous NO in mammalian cells. Thisinvention thus shows that NOSs will regulate both their own targetinteractions and also those of other proteins within signalingassemblies. It is important to emphasize that current proteomic screensare performed in the absence of NO, whereas NO is ubiquitous inmammalian cells. Accordingly, broader application of the methodspresented here should lead to the discovery of a large array of novelinteractions, to new biological functions, and ultimately to a moreaccurate description of the human proteome.

Compounds contemplated for use in the invention include both reactiveoxygen species (ROS) and reactive nitrogen species (RNS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates NO-dependent interactions revealed in a modifiedyeast two-hybrid system.

FIG. 2 illustrates that endogenous NO mediates reversible interactionsbetween acid sphingomyelinase (ASM) and caspase-3 in mammalian cells.

FIG. 3 illustrates an in vitro assay for determining NO-inducedprotein-protein interactions.

FIG. 4 illustrates dynamic NO-regulated interactions between NOsynthases and caspase-3 in vivo.

FIG. 5 illustrates NO-dependent interaction of apoptosis inducing factor(AIF) with macrophage inflammatory protein-1 alpha (MIP-1α).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “protein” and “protein complex” are used synonymouslywith “polypeptide” and “polypeptide complex”. A “purified” polypeptide,protein or biologically active portion thereof is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the polypeptide is derived, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of protein in which the protein is separated fromcellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of polypeptide complexhaving less than about 30% (by dry weight) of non-complex proteins (alsoreferred to herein as a “contaminating protein”), more preferably lessthan about 20% of contaminating protein, still more preferably less thanabout 10% of contaminating protein, and most preferably less than about5% non-complex protein. When the polypeptide or complex is recombinantlyproduced, it is also preferably substantially free of culture medium,e.g., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

As used herein, the term “analogous protein complex” refers to thecomparison of protein complexes between two different cells. A proteincomplex found in cell “A” would be analogous to a protein complex incell “B” if the two protein complexes are identical. The “analogousprotein complex” may be present to a lesser degree in cell “A” versescell “B” or it may be present in cell “A” and not present in cell “B”.

As used herein, the term “lesser degree” refers to the extent or measureof a difference in a protein complex between a first and second cell.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, e.g.,molecules that contain an antigen binding site that specifically binds(immunoreacts with) an antigen, such as a polypeptide complex. Suchantibodies include, e.g., polyclonal, monoclonal, chimeric, singlechain, Fab and F(ab′)2 fragments, and an Fab expression library. Inspecific embodiments, antibodies are generated against human orthologcomplexes.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of a polypeptide complex. A monoclonalantibody composition thus typically displays a single binding affinityfor a particular protein with which it immunoreacts.

As used herein, “modulate” is meant to refer to an increase or decreasein the rate at which a complex is assembled or dissembled, or toincrease or decrease the stability of an assembled complex. Thus, anagent can be tested for its ability to disrupt a complex, or to promoteformation or stability of a complex.

As used herein, the term “derivative” or “derived” refers to a chemicalsubstance, for example a truncated protein or peptide, relatedstructurally to another substance and theoretically derivable from it.

As used herein, the term “region”, as in protein region, refers to anindefinite number of amino acids in a defined area of a parent protein.

As used herein, the term “physiologically levels” refer to acharacteristic of or appropriate to an organism's healthy or normalfunctioning. As used herein, the term “physiologically compatible”refers to a solution or substance, for example media, that can beutilized to mimic an organism's healthy or normal environment. For invivo use, the physiological compatible solution may includepharmaceutically acceptable carriers, excipients, adjuvants,stabilizers, and vehicles. The composition may be in solid, liquid, gel,or aerosol form.

The terms “cell culture medium” and “culture medium” refer to a nutrientsolution used for growing cells that typically provides at least onecomponent from one or more of the following categories: 1) an energysource, usually in the form of a carbohydrate such as glucose; 2) allessential amino acids, and usually the basic set of twenty amino acidsplus cysteine; 3) vitamins and/or other organic compounds required atlow concentrations; 4) free fatty acids; and 5) trace elements, wheretrace elements are defined as inorganic compounds or naturally-occurringelements that are typically required at very low concentrations, usuallyin the micromolar range.

For mammalian cells, the cell culture medium is generally “serum free”when the medium is essentially free of serum from any mammalian source(e.g. fetal bovine serum (FBS)). By “essentially free” is meant that thecell culture medium comprises between about 0-5% serum, preferablybetween about 0-1% serum, and most preferably between about 0-0.1%serum. Advantageously, serum-free “defined” medium can be used, whereinthe identity and concentration of each of the components in the mediumis known (ie., an undefined component such as bovine pituitary extract(BPE) is not present in the culture medium).

As defined herein “specific binding” refers to the ability of twoproteins, peptides or an antibody and antigen to interact.

As used herein, the term “disrupt” refers to the displacement of atleast one polypeptide from a complex of at least two polypeptides.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

As used herein “ROS” refers to, for example, hydrogen peroxide,superoxide, hypochlorite ion and hydroxyl radical.

As used herein, an “RNS” refers to, for example, nitric oxide or nitricdioxide. In a preferred embodiment, the RNS is nitric oxide andcompounds that release nitric oxide or otherwise directly or indirectlydeliver or transfer nitric oxide in vivo to a site of its activity, suchas on a cell membrane.

As used here, the term “nitric oxide” encompasses uncharged nitric oxide(NO) and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO wherein X is a nitric oxide releasing, delivering ortransferring moiety, include any and all such compounds which providenitric oxide to its intended site of action in a form active for theirintended purpose.

As used herein, the term “nitric oxide adducts” encompasses any of suchnitric oxide releasing, delivering or transferring compounds, including,for example, S-nitrosothiols, S-nitroso amino acids,S-nitroso-polypeptides, and nitrosoamines. It is contemplated that anyor all of these “nitric oxide adducts” can be mono- or poly-nitrosylatedat a variety of naturally susceptible or artificially provided bindingsites for nitric oxide. In a preferred embodiment, the NO adduct isdiethylenetriamine-NO (DETA-NO).

The headings for the subsequent sections are provided for organizationalpurpose only. They are not to be considered limiting.

Identification of Protein Interactions Regulated by Redox State ModifierMolecules

This invention provides novel methods for identifying proteininteractions that are regulated by the redox state of the cell in whichthey occur and may, therefore, be regulated by a “redox state modifiermolecule” (RSMM). An RSMM is able to modify the redox state of a cell.In general, modification of redox state will be a change of the redoxstate as compared to normal physiological conditions. Specifically, thisinvention provides a method for identifying a protein complex whoseformation is inhibited by an RSMM (“RSMM inhibited protein complex”); amethod for identifying a protein complex whose formation is induced byan RSMM (“RSMM induced protein complex”); a method for identifying anagent capable of inhibiting RSMM induction of a protein complex; and amethod for identifying an agent capable of inhibiting an RSMM inhibitionof protein complex formation.

(a) RSMMs

In many cases, RSMMs and concentrations thereof associated with aparticular pathophysiologic process are already known. For example,superoxide is associated with the classical model of1-metal-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson'sdisease and nitration is implicated in depletion of dopamine.Hypotensive shock is produced by NO generated by upregulation of nitricoxide synthase. Hyperglycemia (30 millimolar D-glucose) producesselective oxidative stress within the mitochondria. The source of thisredox state modification is nitric oxide synthase and the molecule itproduces in this case is superoxide.

Alterations of the concentrations of various physiological conditionscan cause changes in redox states, which can lead to pathophysiologicalprocesses. For example, variations in glucose concentration have beenlinked to diabetes and ischemia reperfusion injury. pH variation isassociated with mitochondria during apoptosis, with ischemic areas andwith abscess or infected area. Where a pathophysiological process is notknown to involve pH variation from non-pathophysiological state, thiscan be screened for by dyes or electrodes. Disorders are known which areassociated with alteration of metal presence from normal. For example,acrodermatitis enteropathatica results from malabsorption of zinc, andWilson's disease involves copper toxicosis. There is a secondary effectof redox state modification where the alteration of metal amount affectsredox state. For example, copper ions are well known to participate inredox reactions and zinc and cadmium influence the redox state of cells,e.g., by chelating thiol.

Changes in redox states also involve the alteration of an NADH ratio.NADH concentration is altered, e.g., in the case of sleep disordersrelated to circadian rhythms. Alteration of NADH levels can be produced,for example, by knockout of lactate dehydrogenase (LDH), e.g., in yeastcells in a yeast two-hybrid determination.

This invention also provides methods of inducing novel protein-proteininteractions as well as inhibiting an otherwise present protein-proteininteraction. The term protein-protein interaction refers to any type ofmeaningful interaction that proteins are capable of (protein-RNA,receptor-ligand, etc.), and these interactions mediate most cellularprocesses. The strength of binding between two proteins is typicallydescribed by the dissociation constant (K_(d)) and can be determined bymany methods. Generally, interacting polypeptides form heterodimers witha dissociation constant (K_(d)) of at least about 1×10³ M⁻¹, usually atleast 1×10⁴ M⁻¹, typically at least 1×10⁵ M⁻¹, preferably at least 1×10⁶M⁻¹ to 1×10⁷ M⁻¹ or more, under suitable physiological conditions.

In one embodiment, endothelial cell mitochondrial protein-proteininteractions are determined in the presence of superoxide generatingsystems, e.g., by adding an herbicide, such as paraquat, or bytransfecting yeast with nitric oxide synthase and then adding paraquat.

In one embodiment, redox state conditions include physiological pO₂,physiological concentrations of NO, physiological levels ofnitrosothiols, very low levels of reactive oxygen species, and reducingconditions.

The oxygen levels utilized are much lower than the current level, whichis conventionally used, namely the concentration of oxygen in air, roomair having a pO₂ of 150 mm Hg. The oxygen concentrations utilized arepreferably those in the tissue or organ or blood perfusing through forthe physiological process. This concentration varies widely. Forexample, alveolar pO₂ is 100 mm Hg, skeletal muscle pO₂ ranges from 10to 30 mm Hg and exercising muscle is still lower and the pO₂ in thevillus (a loop in the small intestine) is close to zero. Moreover, whilephysiological pO₂ is considered to be ˜30 mm Hg, the pO₂ on running is˜5 mm Hg and rises above 30 mm Hg on abrupt stopping of running, and thepO₂ in the brain associated with thinking is 10-20 mm Hg. Thus, a rangeof oxygen concentrations are considered suitable for the physiologicaldetermination but the oxygen concentration used should be lower thanthat of room air and is preferably below 100 mm Hg. The physiologicalconcentration of NO utilized is nanomolar to submicromolarconcentration. In one embodiment, NO is utilized in a concentration of10 nM to 1 μM. The physiological levels of nitrosothiols utilized range,for example, from 0.01 uM to 10 μM. The physiological levels of reactiveoxygen species utilized range, for example, from 10⁻¹⁰ M to 10⁻⁶ M.Reducing conditions are also not present in conventional determinationswhich are carried out in room air. Reducing conditions can be provided,for example, by adding thiols or NAD(P)H, lowering 0₂ concentration,adding chelating metals, adding physiological levels of ascorbate, e.g.,to provide a concentration of 100 μM, or adding vitamin E. Theappropriate redox state conditions can be effected by adding moleculesto the experiment or by controlling the environment of thedetermination.

In another embodiment, the RSMM is produced by stimulation of an enzyme.The stimulation may be provided by addition of calcium/L-arginine,bradykinin, EGF or other cytokine, growth factor, neuroheumal, ordevelopmental stimulus or activator of a G protein coupled receptor. TheRSMM may be produced from an RSMM-generating enzyme. In one embodiment,the RSMM-generating enzyme is produced from a recombinantRSMM-generating enzyme vector. In a preferred embodiment, theRSMM-generating enzyme is NO synthase, NADPH oxidase, or aconstitutively active rac G-protein.

RSMMs generated by enzymes include, for example, superoxide, peroxides(e.g., hydrogen peroxide), alkoxides, sulfoxides, brominating species,chlorinating species, nitrosating molecules (e.g., NO and RSNO where Ris, for example, amino acid, peptide or protein), and nitratingmolecules (e.g., peroxynitrite) and NO— generating molecules (e.g.,Angeli's salt). These are generated relatively specifically in differentdiseases to different extents and/or in different subcellularcompartments and the means exist to measure these with standardspectroscopic, immunological, electrochemical, chemical and photolyticapproaches. Other RSMMs include enzymes that regulate glutathione, NADHand flavin levels and whose activities can be pharmacologically orgenetically altered. Another important RSMM is O₂ in concentration inaffected body tissue. Body tissue oxygen concentrations are much lowerthan the concentration of oxygen in air, room air having a pO₂ of 150 mmHg. For example, tumors can have a pO₂ in the range of 10 mm Hg and apO₂ in the case of oxygen induced reperfusion can be 80 mm Hg.

In a preferred embodiment, RSMMs are those compounds produced in vivothat are often characteristic of a pathophysiological process and which,because of their presence and/or concentration affect the redox state.In a more preferred embodiment the RSMM is a compound which directlyaffects the redox state. In a more preferred embodiment, the RSMM isselected from the group consisting of nitric oxide, nitric dioxide,dinitrogen trixide, dinitrogen tetraoxide, S-nitrosothiol, nitroxylanion, HNO, nitrite, nitrate, C—, N, O, S or metal-nitroso or nitrocompounds, hydrogen peroxide, peroxynitrite, other peroxides, alkoxides,superoxide, hypochlorite ion, hydroxyl radical and physiological pO₂.

In another preferred embodiment, the pO₂ is in a range from about 5 toabout 100 mm Hg. In a more preferred embodiment, the range of pO₂ isfrom about 10 to about 50 mm Hg. In a most preferred embodiment, therange of pO₂ is from about 10 to about 30 mm Hg.

In another embodiment, the RSMM is an NO adduct. In one embodiment, thenitric oxide adducts are selected from the group of the S-nitrosothiols,which are compounds that include at least one —S—NO group. Suchcompounds include S-nitroso-polypeptides (the term “polypeptide”includes proteins and also polyamino acids that do not possess anascertained biological function, and derivatives thereof);S-nitrosylated amino acids (including natural and synthetic amino acidsand their stereoisomers and racemic mixtures and derivatives thereof);S-nitrosated sugars, S-nitrosated-modified and unmodifiedoligonucleotides (preferably of at least 5, and more particularly 5-200,nucleotides); and an S-nitrosated hydrocarbon where the hydrocarbon canbe a branched or unbranched, and saturated or unsaturated aliphatichydrocarbon, or an aromatic hydrocarbon; S-nitroso hydrocarbons havingone or more substituent groups in addition to the S-nitroso group; andheterocyclic compounds. S-nitrosothiols and the methods for preparingthem are described in U.S. patent application Ser. No. 07/943,834, filedSep. 14, 1992, Oae et al., Org. Prep. Proc. Int., 15(3):165-198, 1983;Loscalzo et al., J. Pharmacol. Exp. Ther., 249(3):726729, 1989, andKowaluk et al., J. Pharmacol. E. Ther., 256:1256-1264, 1990, all ofwhich are incorporated in their entirety by reference.

In a preferred embodiment, the NO adduct is selected from the groupconsisting of DETA-NO, S-nitrosothiol, SIN-1, angeli's salt, S-nitrosoamino acids, S-nitroso-polypeptides, and nitrosoamines.

(b) RSMM Modified Media and Cell Culture

In one aspect, the invention provides a media for culturing a yeast cellcomprising: (a) a physiologically compatible solution; and (b) at leastone RSMM, in a concentration sufficient to modulate a redox reaction.

In yet another embodiment, the media is a liquid media. The media couldbe a semisolid media. In one embodiment, the media comprises betweenabout 0.3% and about 10% of a solidifying agent. The solidifying agentcould be agar or agarose. In a preferred embodiment, the media furthercomprises a substrate. In a preferred embodiment, the substrate is IPTGor ONPG. The media may also further comprise an inducer. In oneembodiment, the inducer is X-gal. In another embodiment, the media islyophilized. In yet another embodiment, the concentration of the RSMM isbetween 100 nM and 1 μM and the RSMM is present for a period of 24hours. In a preferred embodiment, the yeast cell is S. cerevisiae. In afurther embodiment, the yeast cell does not express a functionalflavohemoglobin gene.

This invention provides a method for culturing cells comprising:providing a media comprising an RSMM adduct; and culturing the cell inthe media. In a preferred embodiment, the cell is a yeast cell lackingthe flavohemoglobin gene. Flavohemoglobins are monomeric proteinscontaining one heme and one FAD as prosthetic groups and NADH as aco-factor. The primary sequence and the characterization of the proteinare known for several prokaryotic and eukaryotic organisms.Flavohemoglobins act as NO denitrosylases, which convert NO and O₂ toNO₃ ⁻. Therefore, by deleting the flavohemoglobin gene, the yeast areunable to consume NO.

In a preferred embodiment of the invention, the host cell is cultured ina media comprising a physiologically compatible solution; and at leastone RSMM. The liquid media could be any broth or broth lyophilized intopowders for example, LB broth, YT broth or NZY broth (InvitrogenCorporation, Carlsbad, Calif.). In other embodiments, the media is softagar or solid agar.

In another aspect, the invention provides a method for culturing a cellin vitro comprising: (a) providing a media comprising at least one RSMM;and (b) culturing said cell in the media. In one embodiment, the RSMM isselected from the group consisting of nitric oxide, nitric dioxide,hydrogen peroxide, superoxide, hypochlorite ion, hydroxyl radical andphysiological pO₂.

In one embodiment, the concentration of the RSMM is between 100 nM to 1μM and the time is a period of 24 hours. In another embodiment, themedia is a liquid media or a semisolid media. In a further embodiment,the media comprises between 0.3% to 10% of a solidifying agent. Thesolidifying agent may be agar or agarose.

(c) Methods

(1) RSMM Induction and Inhibition of Protein-Protein Interactions

In one aspect, the invention provides a method for identifying an RSMMinhibited protein complex comprising: (a) culturing a first cell in amedia comprising an RSMM; (b) culturing a second cell in a media withoutthe RSMM; (c) identifying a first protein complex that exists in thesecond cell; (d) analyzing the first cell to determine the existence ofa protein complex analogous to the first protein complex; wherein theRSMM inhibited protein complex is identified when the analogous proteincomplex does not exist or exists to a lesser degree than the firstprotein complex.

In another aspect, the invention provides a method for identifying anRSMM induced protein complex comprising: (a) culturing a first cell in amedia comprising an RSMM; (b) culturing a second cell in a media withoutthe RSMM; (c) identifying a first protein complex that exists in thefirst cell; (d) analyzing the second cell to determine the existence ofa protein complex analogous to the first protein complex; wherein theRSMM induced protein complex is identified when the analogous proteincomplex does not exist or exists to a lesser degree than the firstprotein complex.

In another aspect, the invention includes a method for identifying anagent capable of inhibiting an RSMM induced protein complex comprising:(a) culturing a first cell in a media comprising an RSMM and a testagent; (b) culturing a second cell in a media without the RSMM; (c)analyzing the first cell to determine the existence of the RSMM inducedprotein complex; wherein the agent is identified when the RSMM inducedprotein complex does not exist or exists to a lesser degree than theprotein complex that exists in the first cell in the absence of the testagent.

In one embodiment, the cell is a mammalian cell. In another embodiment,the cell is a hybrid cell. In a preferred embodiment, the hybrid cell isa yeast cell. In another embodiment, the protein complex includes atleast one fusion protein. The fusion protein may be a protein fused to adetectable marker. In another embodiment, the protein complex includes abait protein and a prey protein.

(2) Agents that Disrupt Protein-Protein Interactions

In another aspect, the invention provides a method of determiningwhether a first protein and a second protein interact in an environmentcomprising physiological levels of at least one RSMM, wherein the methodcomprises: (a) transfecting a host cell with a DNA construct expressinga first protein and a second protein to form a transfected host cell;(b) providing a media that comprises at least one RSMM; (c) culturingthe transfected host cell to co-express the first and second proteinsintracellularly; and (d) detecting whether between the first protein andthe second protein interact.

The proteins for which the interactions are determined are any that areexpressed in the physiological process and can be, for example,expressed in the kind of tissue that is affected by thepathophysiological process being compared to, and/or can be the sameproteins (e.g., the same baits and preys). In a preferred embodiment,these proteins are cell death proteins and cell cycle proteins orderivatives thereof. In a preferred embodiment, proteins associated witha pathophysiological process include the NMDA receptor in stroke orcaspase 3 in apoptosis.

In another aspect, the invention provides a method for identifying acompound capable of modulating a protein-protein interaction comprising:(a) providing a cell culture media containing at least one RSMM; (b)culturing a cell that expresses a first protein and a second protein inthe cell culture media containing at least one RSMM, wherein aninteraction between the first protein and second protein produces afirst detectable signal; (c) contacting the cell with the compoundwherein an interaction between the first protein and the second proteinproduces a second detectable signal, wherein the second detectablesignal being lower than the first detectable signal is an indicationthat the compound is capable of modulating the interaction between thefirst and second protein.

In another aspect, the invention provides a method of detectingdifferences between a protein-protein interaction within a first celland a second cell: (a) culturing the first cell in a media comprising anRSMM; (b) isolating a protein complex from the first cell; and (c)comparing the protein complex to a protein complex from the second cellgrown in media without an RSMM. In one embodiment, the first cell andsecond cell are mammalian cells. In another embodiment, the isolating ofthe protein complex is by immunoprecipitation. The protein complex maycomprise multiple members and at least one member is labeled with adetectable label. In one embodiment, the detectable label is selectedfrom the group consisting of biotin, chemiluminescence, digoxigenin,fluorescence, iodination, kinase, ubiquitin and oligosaccharide.

In one embodiment, the interaction of proteins or protein regions isdetected by a growth assay. In another embodiment, the cell culturemedia comprises an RSMM concentration of 1 nM to 1000 μM. The method mayalso further comprise the step of altering the concentration of the RSMMduring culturing. In one embodiment, the alteration is an increase inthe concentration of the RSMM. In another embodiment, the alteration isa decrease in the concentration of the RSMM. The RSMM may beadministered at a physiological level. In one embodiment, there is morethan one RSMM provided to the cell. In a preferred embodiment, at leastone RSMM is nitric oxide. A growth assay can be performed by utilizing acolorimetric substrate in the media as well as an inducer of thesubstrate. In another embodiment, the media further comprises acolorimetric substrate, such as IPTG, and an inducer, such as Xgal. BothIPTG and Xgal are utilized for detection of lacZ transcription. LacZ isa bacterial gene used as a reporter construct for determination oftransfection efficiency as well as histochemical localization followingtransfection of eukaryotic cells. The lacZ gene product β-galactosidasecatalyzes the hydrolysis of the substrate X-gal to produce a blue colorthat is easily visualized with a microscope.

The invention also provides a method of detecting an interaction betweena first protein and a second protein in an environment comprisingphysiological levels of at least one RSMM, the method comprising: (a)transfecting a host cell with a DNA construct expressing a first proteinand a second protein to form a transfected host cell; (b) providing amedia that comprises at least one RSMM; (c) culturing the transfectedhost cell to co-express the two proteins intracellularly; and (d)detecting an interaction of the two proteins.

The first protein and the second protein may be recombinantly expressed.In one embodiment, the first detectable signal or second detectablesignal is detected by a calorimetric assay. In one embodiment, the firstprotein and the second protein are involved in cell death or cellgrowth. In another embodiment, the first protein and the second proteinare cell division cycle proteins or derivatives thereof.

The invention also provides a method of identifying target proteinsand/or genes in a disease specific manner comprising challenging cellsinvolved in a disease with agent(s) to produce redox state-relatedmodification of proteins and/or lipids that would subsequently mediateprotein modification or provide interactions with proteins that arecharacteristic of the disease. For example, very low pO₂ ischaracteristic of a tumor.

The invention also provides a method of correlating proteininteraction(s) with oxygen tension, comprising determining proteininteraction(s) in the presence of oxygen tension different from that inroom air, e.g., in the presence of oxygen tension less than 150 mmHg.

In one embodiment, the method can be carried out using the conventionalmethods for determining protein-protein interactions, for example,two-hybrid systems, including yeast two-hybrid systems described above,except that the determinations are not carried out in room air but inthe presence of oxygen tension less than that in room air, e.g., at pO₂less than 150 mm Hg. In another embodiment, the oxygen tensions usedpreferably range from 0.1 mm Hg to 145 mm Hg, e.g., from 5 mm Hg to 100mm Hg.

In another embodiment, the method can be carried out, for example, usinghigh throughput screens for proteins, e.g., as described in Fung, E. T.,et al. Current Opinion in Biotechnology 12, 65-69 (2001) and computerbased bioinformatic approaches as described in Fung, E. T., et al,Current Opinion in Biotechnology 12, 65-69 (2001) in the presence of theagent(s) to produce redox state-related modifications of proteins and/orlipids that are characteristic of the disease, to identify specificRSMMs and specific protein and lipid related changes and thereby createredox maps of disease. Such maps can be used to create redox chips thatare specific for diseases such as atherosclerosis or Alzheimer's diseasewhere the samples used in conjunction with the chips can be DNA or RNAor protein material. The agent(s) to produce redox state relatedmodifications are, for example, the RSMMs previously described.Preferably, the methods of the invention are carried out in the presenceof oxygen concentration as determined in the tissue or organs affectedby the disease or in blood perfusing said organs.

The invention also provides a method for screening a candidate compoundfor modulation of a protein-protein interaction comprising: (a) a cellculture media containing at least one RSMM, in an amount sufficient tomodulate a redox reaction; (b) providing a cell that expresses a firstand second protein wherein an interaction between the first and secondprotein produces a detectable signal in the presence of the RSMM; (c)contacting said cell with said candidate compound; and (d) detectingsaid signal to determine an effect of the compound on the proteininteraction.

In a preferred embodiment, a plurality of determinations are carried outfor each set of proteins, with different oxygen tensions being employedin each determination, e.g., using 5 or 10 different oxygen tensionswhere the oxygen tensions employed are in increments of 5 or 10 mm Hg.

This invention also provides a method of identifying a previouslyunknown receptor or orphan receptor or activating ligand, comprisingmeasuring activation of receptor or orphan receptor in the presence ofalteration of redox state of ligand. As previously indicated, generalmethods for identifying receptor, orphan receptor and activating ligandare described in Sakura, T., et al., Cell, 573-585 (1988) and Hare, J.,et al., Nature Medium, 5, 124 1-1242 (1999). This class of method ismodified in the invention herein by screening for receptor or orphanreceptor or activating ligand by carrying out the identifying methods ina series of runs in the presence of a series of redox statemodifications, whereby the receptor and activating ligand are associatedwith particular redox state modifications.

This invention also provides a method of determining epitopes involvedin and/or representing markers of disease, comprising immunolabelingaffected tissue or cells in the presence of redox state changes that arecharacteristic of the disease. General methodology useful in this methodis described in Scherer, P., et al., Nature Biology 16, 58 1-586 (1998).The method of Scherer et al. is modified in the invention herein, incarrying out the determination in the presence of redox statemodifications that are characteristic of the disease.

(3) Yeast Two-Hybrid Systems

In one embodiment, the cell is a yeast cell. In a preferred embodiment,the yeast cell is S. cerevisiae. In yet another embodiment, the yeastcell does not express a functional flavohemoglobin gene.

In one aspect, the invention provides a method for detecting aninteraction between a first test protein and a second test protein, themethod comprising: (a) providing a yeast host cell comprising adetectable gene which expresses a detectable protein when the detectablegene is activated by a transcriptional activation domain when thetranscriptional activation domain is in sufficient proximity to thedetectable gene; (b) providing a first nucleic acid that encodes a firsthybrid protein, the first hybrid protein comprising (i) a DNA-bindingdomain that recognizes a binding site in sufficient proximity to thedetectable gene; and (ii) the first test protein; (c) providing a secondnucleic acid encoding a second hybrid protein, the second hybrid proteincomprising: (i) the transcriptional activation domain; and (ii) thesecond test protein; (d) introducing the first chimeric gene and thesecond chimeric gene into the host cell; (e) culturing said host cell ina media comprising an RSMM in an amount sufficient to modulate a redoxreaction; (f) determining whether the detectable gene has beenexpressed, wherein expression of the detectable gene indicates aninteraction between the first test protein and the second test protein.

In yet another embodiment, the DNA-binding domain and thetranscriptional activation domain are derived from GAL4, GCN4 or ADR1.

The first nucleic acid or the second nucleic acid could be an insertfrom a genomic DNA or cDNA library. In one embodiment, the cDNA libraryis derived from a cell selected from the group consisting of amacrophage, a cytokine activated macrophage, an endothelial cell, amuscle cell or a tumor cell. In another embodiment, the cDNA library isderived from a cell treated with a drug. The drug may be achemotherapeutic drug, for example, cisplatin. In another embodiment,the first hybrid protein and second hybrid protein are recombinantlyexpressed. In a preferred embodiment, the first test protein or secondtest protein is a prey protein. In another preferred embodiment, thefirst test protein or second test protein is a bait protein. In yetanother embodiment, the expression of the detectable gene is visualizedby a colorimetric assay. In one embodiment, the interaction of the firsttest protein and second test protein is determined by yeast two hybridassay detection. In another embodiment, a growth assay is performed onthe yeast host cell in the presence and absence of the RSMM. The firsttest protein and second test protein may be involved in cell death orcell growth. The first test protein and the second test protein may becell division cycle proteins or derivatives thereof. In one embodiment,the yeast host cell is S. cerevisiae. In another embodiment, the yeasthost cell does not express a functional flavohemoglobin gene.

In another aspect, the invention provides a method for detecting aninteraction between a first protein region and a second protein regioncomprising: (a) transfecting a yeast cell with a recombinant reportergene coding for a detectable gene product; (b) transfecting the yeastcell with a first recombinant gene coding for a prey fusion protein, theprey fusion protein comprising a transcriptional enhancer domain andsaid first protein region; (c) transfecting the yeast cell with a secondrecombinant gene coding for a bait fusion protein, the bait fusionprotein comprising a DNA-binding domain which binds a sequence on thereporter gene and the second protein region; and, (d) culturing theyeast cell in a media containing at least one RSMM in an amountsufficient to modulate a redox reaction, wherein reporter geneexpression indicates the interaction between the first protein regionand the second region.

In yet another embodiment, the DNA-binding domain and thetranscriptional activation domain are derived from GAL4, GCN4 or ADR1.The prey fusion protein or the bait fusion protein may be encoded by aninsert from a genomic DNA or cDNA library. In one embodiment, the cDNAlibrary is derived from a cell selected from the group consisting of amacrophage, a cytokine activated macrophage, an endothelial cell, amuscle and a tumor cell. In another embodiment, the cDNA library isderived from a cell treated with a drug. In yet another embodiment, thedrug is a chemotherapeutic drug, for example, cisplatin. The prey fusionprotein and the bait fusion protein may be recombinantly expressed. Thedetectable gene product may be detected by a calorimetric assay. Inanother embodiment, a growth assay on the yeast cell is performed in thepresence and absence of the RSMM. The first and second protein regionsmay be derived from proteins involved in cell death or cell growth. Thefirst and second protein regions may be derived from cell division cycleproteins or derivatives thereof. In one embodiment, the yeast cell is S.cerevisiae. In another embodiment, the yeast cell does not express afunctional flavohemoglobin gene.

In one embodiment, the first protein and second protein may berecombinantly expressed. In one embodiment, the first protein or secondprotein is a prey protein. In a further embodiment, the first protein orsecond protein is a bait protein. The interaction between the firstprotein and second protein may be detected by a calorimetric assay or ayeast two hybrid assay. In another embodiment, this method furthercomprises performing a growth assay on the cell in the presence andabsence of the RSMM. The first protein and second protein may beinvolved in cell death or cell growth. The first protein and secondprotein may be cell division cycle proteins or derivatives thereof.

(d) Purified Protein Complexes

In another aspect, the invention provides a purified complex comprisinga first polypeptide and a second polypeptide, wherein the firstpolypeptide comprises an amino acid sequence selected from an apoptoticsignaling molecule, wherein the second polypeptide comprises an aminoacid sequence of the corresponding sequence selected from the groupconsisting of ASM, IRG, PGM-1, iNOS, nNOS and eNOS, and wherein thefirst polypeptide and the second polypeptide bind only in the presenceof at least one RSMM. In one embodiment, the first polypeptide isselected from the group consisting of caspase-3, caspase-8, caspase-9,Apaf-1, Bcl-2 and AIF. The first polypeptide and second polypeptides maybe labeled.

In one embodiment, the protein complex includes at least one fusionprotein. The fusion protein may be a protein fused to a detectablemarker. In another embodiment, the protein complex includes a baitprotein and a prey protein.

In one aspect, the invention provides a purified complex comprising afirst polypeptide and a second polypeptide, wherein the firstpolypeptide comprises a region of amino acids of an apoptotic signalingmolecule, and wherein the second polypeptide comprises a region of aminoacids of an interacting polypeptide selected from the group consistingof ASM, IRG, PGM-1, iNOS, NNOS and eNOS, and wherein the firstpolypeptide and the second polypeptide bind only in the presence of atleast one RSMM. In one embodiment, the apoptotic signaling molecule iscaspase-3, caspase-8, caspase-9, Apaf-1, Bcl-2 or AIF.

In another aspect, the invention provides a chimeric polypeptidecomprising six or more amino acids of the first polypeptide of thepreviously described purified complex covalently linked to six or moreamino acids of the second polypeptide of the previously describedpurified complex. In another aspect, the invention provides a nucleicacid encoding the chimeric polypeptide. In a further aspect, theinvention provides a vector comprising the nucleic acid previouslydescribed. In yet another aspect, the invention provides a cellcomprising the vector. In another aspect, the invention provides anantibody that specifically binds the purified complex. In oneembodiment, the antibody specifically binds to the purified complex witha higher affinity than it binds to the first or second polypeptide whenthe polypeptides are not complexed.

This invention includes a purified complex that includes two or morepolypeptides. In one embodiment, the invention provides purifiedcomplexes of two or more polypeptides. In a preferred embodiment, thiscomplex is a redox associated polypeptide complex. As used herein, “aredox associated polypeptide complex” is meant to be a polypeptidecomplex that forms due to a redox state modification. One of thepolypeptides includes a polypeptide selected from an apoptotic signalingpeptide. In another embodiment, the first and second polypeptides areselected from the group consisting of caspase-3, caspase-8, caspase-9,Apaf-1, Bcl-2, AIF, ASM, IRG, PGM-1, iNOS, nNOS and eNOS. In someembodiments a first polypeptide is listed as a “bait” polypeptide and asecond polypeptide is denoted as “prey” polypeptide, while in otherembodiments the first polypeptide corresponds to a “prey” polypeptideand the second is a “bait” polypeptide.

In certain embodiments, the first polypeptide is labeled. In otherembodiments, the second polypeptide is labeled, while in someembodiments, both the first and second polypeptides are labeled.Labeling can be performed using any art-recognized method for labelingpolypeptides. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

The invention also includes complexes of two or more polypeptides inwhich at least one of the polypeptides is present as a fragment of acomplex-forming polypeptide according to the invention. For example, oneor more polypeptides may include an amino acid sequence sufficient tobind to its corresponding polypeptide. A binding domain of a given firstpolypeptide can be any number of amino acids sufficient to specificallybind to, and complex with, the corresponding second polypeptide underconditions suitable for complex formation. The binding domain can be theminimal number of amino acids required to retain binding affinity, ormay be a larger fragment or derivative of the polypeptides. Proceduresfor identifying binding domains can be readily identified by one ofordinary skill in the art and the procedures described herein. Forexample, nucleic acid sequences containing various portions of a “bait”protein can be tested in a yeast two hybrid screening assay incombination with a nucleic acid encoding the corresponding “prey”protein.

In other embodiments, the complexes are human ortholog complexes,chimeric complexes, or specific complexes implicated in microbialpathways.

This invention also provides novel complexes of NO-dependent interactingpolypeptides which have not heretofore been shown to interact directly,as well as methods of using these complexes.

(e) Identification of Agents Which Affect RSMMs

In another aspect, the invention provides a method of identifying anagent which disrupts a polypeptide complex, the method comprising: (a)providing the complex; (b) contacting the complex with a test agent; and(c) detecting the presence of a polypeptide displaced from the complex,wherein the presence of the displaced polypeptide indicates the agentdisrupts the complex. In one embodiment, the agent is selected from thegroup consisting of a peptide, a small molecule, a soluble receptor, areceptor agonist and an antibody.

In another aspect, the invention provides a method for identifying anagent which disrupts a polypeptide complex comprising at least oneapoptotic signaling protein or cell cycle protein, the methodcomprising: (a) providing the complex previously described; (b)contacting the complex with a test agent; and (c) detecting the presenceof a polypeptide displaced from the complex, wherein the presence ofdisplaced polypeptide indicates said agent disrupts the complex. In oneembodiment, the agent is selected from the group consisting of apeptide, a small molecule, a soluble receptor, a receptor agonist and anantibody. In a further embodiment, the apoptotic signaling protein isselected from the group consisting of caspase-3, caspase-8, caspase-9,Apaf-1, Bcl-2 and AIF. In yet another embodiment, the cell cycle proteinis selected from the group consisting of cyclin, phosphatase, kinase,oncogenic protein and tumor supressor protein.

In another aspect, the invention provides a method of identifying anagent which inhibits S-nitrosylation comprising: (a) culturing a firstcell capable of S-nitrosylation in a media comprising a candidateinhibitor of S-nitrosylation; (b) culturing a second cell capable ofS-nitrosylation in a media without the candidate inhibitor ofS-nitrosylation wherein the second cell is similar to the first cellexcept for lacking the candidate inhibitor; and (c) comparingS-nitrosylation in both the first cell and the second cell wherein theagent which inhibits S-nitrosylation is identified when S-nitrosylationis less in the first cell than in the second cell. In one embodiment,the S-nitrosylation occurs at an S-nitrosylation consensus sequence. Ina preferred embodiment, the S-nitrosylation consensus sequence is XCY,wherein X and Y are acidic and/or basic amino acids. In anotherpreferred embodiment, wherein S-nitrosylation consensus sequence isKRHDE.

The invention further provides methods of identifying an agent whichmodulates the formation or stability of a redox associated polypeptidecomplex described herein.

In one embodiment, the invention provides a method of identifying anagent that promotes disruption of a redox associated polypeptidecomplex. The method includes providing a polypeptide complex, contactingthe complex with a test agent, and detecting the presence of apolypeptide displaced from the complex. The presence of a displacedpolypeptide indicates the disruption of the complex by the agent. Insome embodiments, the complex contains at least one apoptotic signalingmolecule. In another embodiment, the complex contains at least one cellcycle protein. Agents which disrupt complexes of the invention maypresent novel modulators of cell processes and pathways in which thecomplexes participate.

Any compound or other molecule (or mixture or aggregate thereof) can beused as a test agent. In some embodiments, the agent can be a smallpeptide, or other small molecule produced by e.g., combinatorialsynthetic methods known in the art. In other embodiments, the agent canbe a soluble receptor, receptor agonist or antibody. Disruption of thecomplex by the test agent, e.g. binding of the agent to the complex, canbe determined using art recognized methods, e.g., detection ofpolypeptide using polypeptide-specific antibodies, as described above.Bound agents can alternatively be identified by comparing the relativeelectrophoretic mobility of complexes exposed to the test agent to themobility of complexes that have not been exposed to the test agent.

Agents identified in the screening assays can be further tested fortheir ability to alter and/or modulate cellular functions, particularlythose functions in which the complex has been implicated. Thesefunctions include, e.g., control of cell-cycle progression; regulationof transcription; or the control of intracellular signal transduction.

In another embodiment, the invention provides methods for inhibiting theinteraction of a polypeptide with a ligand, by contacting a complex ofthe protein and the ligand with an agent that disrupts the complex, asdescribed above. In certain embodiments, the polypeptides are apoptosissignaling proteins or cell cycle proteins. Inhibition of complexformation allows for modulation of cellular functions and pathways inwhich the targeted complexes participate.

Polypeptides of the Invention and Recombinant Techniques

Polypeptides forming the complexes according to the invention can bemade using techniques known in the art. For example, one or more of thepolypeptides in the complex can be chemically synthesized usingart-recognized methods for polypeptide synthesis. These methods arecommon in the art, including synthesis using a peptide synthesizer. See,e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed.Springer-Verlag, 1988; Merrifield, Science 232: 241-247 (1986); Barany,et al, Intl. J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev.Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198(1989).

Alternatively, polypeptides can be made by expressing one or bothpolypeptides from a nucleic acid and allowing the complex to form fromthe expressed polypeptides. Any known nucleic acids that express thepolypeptides, whether yeast or human (or chimerics of thesepolypeptides) can be used, as can vectors and cells expressing thesepolypeptides. Sequences of yeast ORFs and human polypeptides arepublicly available, e.g. at the Saccharomyces Genome Database (SGD) andGenBank (see, e.g. Hudson et al., Genome Res. 7: 1169-1173 (1997). Ifdesired, the complexes can then be recovered and isolated.

Recombinant cells expressing the polypeptide, or a fragment orderivative thereof, may be obtained using methods known in the art, andindividual gene product or complex may be isolated and analyzed (See,e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993). This isachieved by assays that are based upon the physical and/or functionalproperties of the protein or complex. The assays can include, e.g.,radioactive labeling of one or more of the polypeptide complexcomponents, followed by analysis by gel electrophoresis, immunoassay andcross-linking to marker-labeled products. Polypeptide complexes may beisolated and purified by standard methods known in the art (either fromnatural sources or recombinant host cells expressing theproteins/protein complex). These methods can include, e.g., columnchromatography (e.g., ion exchange, affinity, gel exclusion,reverse-phase, high pressure, fast protein liquid, etc), differentialcentrifugation, differential solubility, or similar methods used for thepurification of proteins. In accordance with the present invention,several interactions have presently been identified where one of theinteracting partners is an apoptotic signaling protein. Inhibiting anyof these interactions could lead to the disruption in cell proliferationor cell death.

As described above, certain embodiments of these complexes contain thebinding domains of these polypeptides, while other embodiments containconservative variants of these polypeptides.

In a further aspect, the invention provides a chimeric polypeptide thatincludes sequences of two interacting proteins according to theinvention. The interacting proteins can be, e.g., the interactingprotein pairs disclosed in the Examples section below. Also included arechimeric polypeptides including multimers, e.g., sequences from two ormore pairs of interacting proteins. The chimeric polypeptide includes aregion of a first protein covalently linked, e.g. via peptide bond, to aregion of a second protein. In certain embodiments, the second proteinis a species ortholog of the first protein. In the preferredembodiments, the chimeric polypeptide contains regions of first andsecond human proteins. In some embodiments, the chimeric polypeptide(s)of the complex include(s) six or more amino acids of a first proteincovalently linked to six or more amino acids of a second protein. Inother embodiments, the chimeric polypeptide includes at least onebinding domain of a first or second protein.

Preferably, the chimeric polypeptide includes a region of amino acids ofthe first polypeptide able to bind to a second polypeptide.Alternatively, or in addition, the chimeric polypeptide includes aregion of amino acids of the second polypeptide able to bind to thefirst polypeptide.

Nucleic acid encoding the chimeric polypeptide, as well as vectors andcells containing these nucleic acids, are within the scope of thepresent invention. The chimeric polypeptides can be constructed byexpressing nucleic acids encoding chimeric polypeptides usingrecombinant methods, described above, then recovering the chimericpolypeptides, or by chemically synthesizing the chimeric polypeptides.Host-vector systems that can be used to express chimeric polypeptidesinclude, e.g.: (i) mammalian cell systems which are infected withvaccinia virus, adenovirus; (ii) insect cell systems infected withbaculovirus; (iii) yeast containing yeast vectors or (iv) bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.Depending upon the host-vector system utilized, any one of a number ofsuitable transcription and translation elements may be used.

The expression of the specific proteins may be controlled by anypromoter/enhancer known in the art including, e.g.: (i) the SV40 earlypromoter (see e.g., Bernoist & Chambon, Nature 290: 304-310 (1981));(ii) the promoter contained within the 3′-terminus long terminal repeatof Rous Sarcoma Virus (see e.g., Yamamoto, et al., Cell 22: 787-797(1980)); (iii) the Herpesvirus thymidine kinase promoter (see e.g.,Wagner, et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)); (iv)the regulatory sequences of the metallothionein gene (see e.g.,Brinster, et al., Nature 296: 39-42 (1982)); (v) prokaryotic expressionvectors such as the β-lactamase promoter (see e.g., Villa-Kamaroff, etal., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)); (vi) the tacpromoter (see e.g., DeBoer, et al., Proc. Natl. Acad. Sci. USA 80: 21-25(1983)).

Plant promoter/enhancer sequences within plant expression vectors mayalso be utilized including, e.g.: (i) the nopaline synthetase promoter(see e.g., Herrar-Estrella, et al., Nature 303: 209-213 (1984)); (ii)the cauliflower mosaic virus 35S RNA promoter (see e.g., Garder, et al.,Nuc. Acids Res. 9: 2871 (1981)) and (iii) the promoter of thephotosynthetic enzyme ribulose bisphosphate carboxylase (see e.g.,Herrera-Estrella, et al., Nature 310: 115-120 (1984)).

Promoter/enhancer elements from yeast and other fungi (e.g., the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter), as well as the followinganimal transcriptional control regions, which possess tissue specificityand have been used in transgenic animals, may be utilized in theproduction of proteins of the present invention.

Other animal transcriptional control sequences derived from animalsinclude, e.g.: (i) the insulin gene control region active withinpancreatic β-cells (see e.g., Hanahan, et al., Nature 315: 115-122(1985)); (ii) the immunoglobulin gene control region active withinlymphoid cells (see e.g., Grosschedl, et al., Cell 38: 647-658 (1984));(iii) the albumin gene control region active within liver (see e.g.,Pinckert, et al., Genes and Devel. 1: 268-276 (1987)); (iv) the myelinbasic protein gene control region active within brain oligodendrocytecells (see e.g., Readhead, et al., Cell 48: 703-712 (1987)); and (v) thegonadotrophin-releasing hormone gene control region active within thehypothalamus (see e.g., Mason, et al., Science 234: 1372-1378 (1986)).

The vector may include a promoter operably-linked to nucleic acidsequences which encode a chimeric polypeptide, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). A host cell strain may be selected whichmodulates the expression of chimeric sequences, or modifies/processesthe expressed proteins in a desired manner. Moreover, different hostcells possess characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, phosphorylation, and the like) of expressed proteins.Appropriate cell lines or host systems may thus be chosen to ensure thedesired modification and processing of the foreign protein is achieved.For example, protein expression within a bacterial system can be used toproduce an unglycosylated core protein; whereas expression withinmammalian cells ensures “native” glycosylation of a heterologousprotein.

Antibodies

The invention further provides antibodies and antibody fragments (suchas Fab or F(ab′)2 fragments) that bind specifically to the complexesdescribed herein. By “specifically binds” is meant an antibody thatrecognizes and binds to a particular polypeptide complex of theinvention, but which does not substantially recognize or bind to othermolecules in a sample, or to any of the polypeptides of the complex whenthose polypeptides are not complexed.

For example, a purified complex, or a portion, variant, or fragmentthereof, can be used as an immunogen to generate antibodies thatspecifically bind the complex using standard techniques for polyclonaland monoclonal antibody preparation.

A full-length polypeptide complex can be used, if desired.Alternatively, the invention provides antigenic fragments of polypeptidecomplexes for use as immunogens. In some embodiments, the antigeniccomplex fragment includes at least 6, 8, 10, 15, 20, or 30 or more aminoacid residues of a polypeptide. In one embodiment, epitopes encompassedby the antigenic peptide include the binding domains of thepolypeptides, or are located on the surface of the protein, e.g.,hydrophilic regions.

If desired, peptides containing antigenic regions can be selected usinghydropathy plots showing regions of hydrophilicity and hydrophobicity.These plots may be generated by any method well known in the art,including, for example, the Kyte Doolittle or the Hopp Woods methods,either with or without Fourier transformation. See, e.g., Hopp andWoods, Proc. Nat. Acad. Sci. USA 78:3824-3828 (1981); Kyte andDoolittle, J. Mol. Biol. 157:105-142 (1982).

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies. For example, for the productionof polyclonal antibodies, various suitable host animals (e.g., rabbit,goat, mouse or other mammal) may be immunized by injection with thenative protein, or a synthetic variant thereof, or a derivative of theforegoing. An appropriate immunogenic preparation can contain, forexample, a recombinantly expressed polypeptide complex. Alternatively,the immunogenic polypeptides or complex may be chemically synthesized,as previously discussed. The preparation can further include anadjuvant. Various adjuvants used to increase the immunological responseinclude, e.g., Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), human adjuvants such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. If desired,the antibody molecules directed against the complex can be isolated fromthe mammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.

For preparation of monoclonal antibodies directed towards a particularcomplex, or polypeptide, any technique that provides for the productionof antibody molecules by continuous cell line culture may be utilized.Such techniques include, e.g., the hybridoma technique (see Kohler &Milstein, Nature 256: 495-497 (1975)); the trioma technique; the humanB-cell hybridoma technique (see Kozbor, et al., Immunol Today 4: 72(1983)); and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole, et al., In: Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., (1985) pp. 77-96). If desired, humanmonoclonal antibodies may be prepared by using human hybridomas (seeCote, et al., Proc. Natl. Acad. Sci. USA 80: 2026-2030 (1983)) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., In: Monoclonal Antibodies and Cancer Therapy, supra).

Methods can be adapted for the construction of Fab expression libraries(see e.g., Huse, et al., Science 246: 1275-1281 (1989)) to allow rapidand effective identification of monoclonal Fab fragments with thedesired specificity for the desired protein or derivatives, fragments,analogs or homologs thereof. Non-human antibodies can be “humanized” bytechniques well known in the art. See e.g., U.S. Pat. No. 5,225,539.Antibody fragments that contain the idiotypes to a polypeptide orpolypeptide complex may be produced by techniques known in the artincluding, e.g.: (i) an F(ab′)2 fragment produced by pepsin digestion ofan antibody molecule; (ii) an Fab fragment generated by reducing thedisulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragmentgenerated by the treatment of the antibody molecule with papain and areducing agent and (iv) Fv fragments.

Chimeric and humanized monoclonal antibodies against the polypeptidecomplexes, or polypeptides, described herein are also within the scopeof the invention, and can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCTInternational Application No. PCT/US86/02269; European PatentApplication No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al., Science 240: 1041-1043 (1988); Liu et al.,Proc. Nat. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol.139: 3521-3526 (1987); Sun et al., Proc. Nat. Acad. Sci. USA 84: 214-218(1987); Nishimura et al., Cancer Res. 47: 999-1005 (1987); Wood et al.,Nature 314: 446-449 (1985); Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988); Morrison, Science 229: 1202-1207 (1985); Oi et al.,BioTechniques 4: 214 (1986); U.S. Pat. No. 5,225,539; Jones et al.,Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534 (1988);and Beidler et al., J. Immunol. 141: 4053-4060 (1988).

Methods for the screening of antibodies that possess the desiredspecificity include, e.g., enzyme-linked immunosorbent assay (ELISA) andother immunologically-mediated techniques known within the art. Forexample, selection of antibodies that are specific to a particulardomain of a polypeptide complex is facilitated by generation ofhybridomas that bind to the complex, or fragment thereof, possessingsuch a domain.

In certain embodiments of the invention, antibodies specific for thepolypeptide complexes described herein may be used in various methods,such as detection of complex, and identification of agents which disruptcomplexes. These methods are described in more detail, below. Detectioncan be facilitated by coupling (e.g., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Polypeptide complex-specific, or polypeptide-specific antibodies, canalso be used to isolate complexes using standard techniques, such asaffinity chromatography or immunoprecipitation. Thus, the antibodiesdisclosed herein can facilitate the purification of specific polypeptidecomplexes from cells, as well as recombinantly produced complexesexpressed in host cells.

Pharmaceutical Compositions, Therapeutic or Diagnostic Uses and Kits

The invention further provides pharmaceutical compositions of purifiedcomplexes suitable for administration to a subject, most preferably, ahuman, in the treatment of disorders involving altered levels of suchcomplexes. Such preparations include a therapeutically-effective amountof a complex, and a pharmaceutically acceptable carrier.

The therapeutic amount of a complex which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and may be determined by standardclinical techniques by those of average skill within the art. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theoverall seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. However, suitable dosage ranges for intravenousadministration of the complexes of the present invention are generallyabout 20-500 micrograms (μg) of active compound per kilogram (Kg) bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems. Suppositories generally containactive ingredient in the range of 0.5% to 10% by weight; oralformulations preferably contain 10% to 95% active ingredient.

Various delivery systems are known and can be used to administer apharmaceutical preparation of a complex of the invention including,e.g.: (i) encapsulation in liposomes, microparticles, microcapsules;(ii) recombinant cells capable of expressing the polypeptides of thecomplex; (iii) receptor-mediated endocytosis (see, e.g., Wu et al., J.Biol. Chem. 262: 4429-4432 (1987)); (iv) construction of a nucleic acidencoding the polypeptides of the complex as part of a retroviral orother vector, and the like.

Methods of administration include, e.g., intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The pharmaceutical preparations of the present inventionmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically-active agents.Administration can be systemic or local. In addition, it may beadvantageous to administer the pharmaceutical preparation into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection. Intraventricular injection may be facilitatedby an intraventricular catheter attached to a reservoir (e.g., an Ommayareservoir). Pulmonary administration may also be employed by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. It mayalso be desirable to administer the pharmaceutical preparation locallyto the area in need of treatment; this may be achieved by, for example,and not by way of limitation, local infusion during surgery, topicalapplication, by injection, by means of a catheter, by means of asuppository, or by means of an implant. In a specific embodiment,administration may be by direct injection at the site (or former site)of a malignant tumor or neoplastic or pre-neoplastic tissue.

Alternatively, pharmaceutical preparations of the invention may bedelivered in a vesicle, in particular a liposome, (see, e.g., Langer,Science 249:1527-1533 (1990)) or via a controlled release systemincluding, e.g., a delivery pump (see, e.g., Saudek, et al., New Engl.J. Med. 321: 574 (1989) and a semi-permeable polymeric material (see,e.g., Howard, et al., J. Neurosurg. 71: 105 (1989)). Additionally, thecontrolled release system can be placed in proximity of the therapeutictarget (e.g., the brain), thus requiring only a fraction of the systemicdose. See, e.g., Goodson, In: Medical Applications of ControlledRelease, 1984 (CRC Press, Bocca Raton, Fla.).

In another embodiment, the invention provides a method for identifying apolypeptide complex in a subject. The method includes the steps ofproviding a biological sample from the subject, detecting, if present,the level of polypeptide complex. In some embodiments, the complexincludes a first polypeptide (a “bait” polypeptide) and a secondpolypeptide (“prey” polypeptide) selected. Any suitable biologicalsample potentially containing the complex may be employed, e.g. blood,urine, cerebral-spinal fluid, plasma, etc. Complexes may be detected by,e.g., using complex-specific antibodies as described above. The methodprovides for diagnostic screening, including in the clinical setting,using, e.g., the kits described above.

In still another embodiment, the present invention provides methods fordetecting a polypeptide in a biological sample, by providing abiological sample containing the polypeptide, contacting the sample witha corresponding polypeptide to form a complex under suitable conditions,and detecting the presence of the complex. A complex will form if thesample does, indeed, contain the first polypeptide. In some embodiments,the polypeptide being detecting is a “prey” protein selected from thepolypeptides caspase-3, caspase-8, caspase-9, Apaf-1, Bcl-2 and AIF, andis detected by complexing with the corresponding “bait” protein, suchas, ASM, IRG, PGM-1, iNOS, NNOS and eNOS. Conversely, in otherembodiments the polypeptide being detected is the “bait” protein.Alternatively, a yeast “bait” or “prey” ortholog may be employed to forma chimeric complex with the polypeptide in the biological sample.

In still another embodiment, the invention provides methods for removinga first polypeptide from a biological sample by contacting thebiological sample with the corresponding second peptide to form acomplex under conditions suitable for such formation. The complex isthen removed from the sample, effectively removing the firstpolypeptide. As with the methods of detecting polypeptide describedabove, the polypeptide being removed may be either a “bait” or “prey”protein, and the second corresponding polypeptide used to remove it maybe either a yeast or human ortholog polypeptide.

Methods of determining altered expression of a polypeptide in a subject,e.g. for diagnostic purposes, are also provided by the invention.Altered expression of proteins involved in cell processes and pathwayscan lead to deleterious effects in the subject. Altered expression of apolypeptide in a given pathway leads to altered formation of complexeswhich include the polypeptide, hence providing a means for indirectdetection of the polypeptide level. The method involves providing abiological sample from a subject, measuring the level of a polypeptidecomplex of the invention in the sample, and comparing the level to thelevel of complex in a reference sample having known polypeptideexpression. A higher or lower complex level in the sample versus thereference indicates altered expression of either of the polypeptidesthat forms the complex. The detection of altered expression of apolypeptide can be use to diagnose a given disease state, and or used toidentify a subject with a predisposition for a disease state. Anysuitable reference sample may be employed, but preferably the testsample and the reference sample are derived from the same medium, e.g.both are urine, etc. The reference sample should be suitablyrepresentative of the level polypeptide expressed in a controlpopulation.

The invention further provides methods for treating or preventing adisease or disorder involving altered levels of a redox associatedpolypeptide complex, or polypeptide, disclosed herein, by administeringto a subject a therapeutically-effective amount of at least one moleculethat modulates the function of the complex. As discussed above, alteredlevels of polypeptide complexes described herein may be implicated indisease states resulting from a deviation in normal function of thepathway in which a complex is implicated. In subjects with adeleteriously high level of complex, modulation may consist, forexample, by administering an agent which disrupts the complex, or anagent which does not disrupt, but down-regulates, the functionalactivity of the complex. Alternatively, modulation in subjects with adeleteriously low level of complex may be achieved by pharmaceuticaladministration of complex, constituent polypeptide, or an agent whichup-regulates the functional activity of complex. Pharmaceuticalpreparations suitable for administration of complex are described above.

The invention also provides a kit to perform the modified two hybridassay. The kit contains a first vector which contains a first chimericgene. This chimeric gene includes a promoter, transcription terminationsignal and a DNA binding domain. The kit also includes a second vectorwhich contains a second chimeric gene. The second chimeric gene alsoincludes a promoter and a transcription termination signal to directtranscription. The second chimeric gene also includes a DNA sequencethat encodes a transcriptional activation domain and a uniquerestriction site(s) to insert a DNA sequence encoding the second testprotein or protein fragment into the vector, in such a manner that thesecond test protein is capable of being expressed as part of a hybridprotein with the transcriptional activation domain. Preferably, theDNA-binding domain of the first hybrid protein and the transcriptionalactivation domain of the second hybrid protein are derived fromtranscriptional activators having separate DNA-binding andtranscriptional activation domains. These separate DNA-binding andtranscriptional activation domains are known to be found in the yeastGAL4 protein and are also known to be found in the yeast GCN4 and ADR1proteins. Many other proteins involved in transcription also haveseparable binding and transcriptional activation domains which make themuseful for the present invention. In another embodiment, the DNA bindingdomain and the transcriptional activation domain may be from differenttranscriptional activators. The second hybrid protein may be encoded ona library of plasmids that contain genomic, cDNA or syntheticallygenerated DNA sequences fused to the DNA sequence encoding thetranscriptional activation domain.

The second vector further includes a means for replicating itself in thehost cell and in bacteria. The second vector also includes a secondmarker gene, the expression of which in the host cell permits selectionof cells containing the second marker gene from cells that do notcontain the second marker gene.

The kit includes a host cell, preferably a yeast strain of Saccharomycescerevisiae or Schizosaccharomyces pombe. The host cell contains thedetectable gene having a binding site for the DNA-binding domain of thefirst hybrid protein. The binding site is positioned so that thedetectable gene expresses a detectable protein when the detectable geneis activated by the transcriptional activation domain encoded by thesecond vector. Activation of the detectable gene is possible when thetranscriptional activation domain is in sufficient proximity to thedetectable gene. The host cell, by itself, is incapable of expressing aprotein having a function of the first marker gene, the second markergene, the DNA-binding domain, or the transcriptional activation domain.

Accordingly in using the kit, the interaction of the first test proteinand the second test protein in the host cell causes a measurably greaterexpression of the detectable gene than when the DNA-binding domain andthe transcriptional activation domain are present, in the absence of aninteraction between the first test protein and the second test protein.The detectable gene may encode an enzyme or other product that can bereadily measured. Such measurable activity may include the ability ofthe cell to grow only when the marker gene is transcribed, or thepresence of detectable enzyme activity only when the marker gene istranscribed. Various other markers are well known within the skill ofworkers in the art.

The cells containing the two hybrid proteins are incubated in anappropriate media, which is optionally supplied with the kit, and theculture is monitored for the measurable activity. A positive test forthis activity is an indication that the first test protein and thesecond test protein have interacted. Such interaction brings theirrespective DNA-binding and transcriptional activation domains intosufficiently close proximity to cause transcription of the marker gene.

In a specific embodiment, the invention provides kits containing areagent, for example, an antibody described above, which canspecifically detect a polypeptide complex, or a constituent polypeptide,described herein. Such kits can contain, for example, reaction vessels,reagents for detecting complex in sample, and reagents for developmentof detected complex, e.g. a secondary antibody coupled to a detectablemarker. The label incorporated into the anti-complex, oranti-polypeptide antibody may include, e.g., a chemiluminescent,enzymatic, fluorescent, calorimetric or radioactive moiety. Kits of thepresent invention may be employed in diagnostic and/or clinicalscreening assays.

Other features of the invention will become apparent in the course ofthe following description of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof. All references, patents and patent applications cited arehereby incorporated by reference in their entirety.

EXAMPLES Example 1 Yeast Two-Hybrid Screening

Methods

Generation of Bait Plasmids

Construction of bait plasmids. A NcoI and XhoI digest of caspase-3(Mannick et al., 1999) was integrated into pAS2-1 (Clontech). The entireopen reading frame of AIF was derived from I.M.A.G.E. clones 1520292 and25192, and sub-cloned into pAS2-1 (between NdeI and EcoRI sites). DNAsequencing confirmed construct identities, and the expression of baitproteins (fused to binding domains (BD)) was verified by immunoblottingwith BD and/or bait-specific monoclonal antibodies.

Modified Yeast

The YHB1 gene was deleted from yeast strain CG-1945 and the absence ofNO consumption activity was verified (Liu et al., 2000). Yeasttwo-hybrid screening was then performed in the CG-1945-Δyhb1 host. Cellswere sequentially transformed with bait (selection in tryptophan(Trp)-deficient medium) and library (selection in Trp-leucine(Leu)-deficient medium) plasmids according to published directions (P.L. Bartel, S. Fields, The Yeast Two-Hybrid System. A. Jacobson, Ed.,Advances in Molecular Biology (Oxford University Press, New York, 1997).Cells containing pairs of interacting proteins were selected by theirgrowth on histidine (His)-deficient medium and by expression ofβ-galactosidase (β-gal) activity.

Preparation of cDNA Library

Poly(A)⁺ mRNA was purified from RAW264.7 cells stimulated with murineIFN-γ (100 units/ml) and bacterial LPS (100 ng/ml) for 0, 2, 4, 6, 8,12, 18 and 24 hrs. The cDNAs were prepared using oligo(dT) and randomprimers, ligated to the EcoRI adaptor (5′-AATTCGCGGCCGCGTCGAC-3′) andcloned into the EcoRI site of pGAD10 (Clontech) prey plasmids. Theaverage insert size was 1.7 kbp and the range of insert size was 0.4˜3.5kbp. The resultant library was amplified once in E. coli to obtain theplasmid cDNA library used for screening.

Yeast Two Hybrid Assay

Yeast (Δyhb1) were transformed with Gal4 BD-caspase-3 plasmid (bait) anda cDNA library (Gal4 AD prey plasmids) derived from activated (NOgenerating) macrophages. Screening was performed as described in themethods below. DETA-NO at less than 300 μM generates NO withoutinhibiting yeast growth (monitored by absorbance at 600 nm). Steadystate NO concentrations are maintained at ˜100 nM-1 μM range for severaldays as measured with an NO electrode (not shown).

Method 1:

Auxotrophic selection was carried out on agar plates (15 cm in diameter)enriched in 50 mM phosphate buffer (pH 7.2; His-Trp-Leu(triple)-deficient buffered medium (TDBM)). Yeast were cultured for 4days at 30° C. in the presence or absence of DETA-NO (40 μl of a 0.3Msolution). Clones that showed at least 3-fold greater growth in DETA-NOwere selected and further analyzed for β-gal activity with ONPG(O-nitrophenyl β-D-galactopyranoside) as substrate. Prey plasmids wereisolated from positive yeast clones and then re-introduced into the baitstrain to confirm bait-prey interactions.

Method 2:

After cells were seeded on 1.5% agar they were covered with 3% lowmelting point agar, which in turn was layered with culture medium. NOdonors (e.g. DETA-NO; 300 μM final concentration) were added to theliquid layer every 24 hours. Colonies are grown for 4 days as describedin ref 9.

Method 3:

Auxotrophic selection was carried out in TDBM. Transformation with thecDNA library was followed by overnight growth in medium deficient intryptophan and leucine. Transformants were then grown for 3 days inHis-deficient medium supplemented with DETA-NO (typically 200 μM finalconcentration). The plasmid DNA was harvested and transformed into E.coli. Individual clones were isolated, retransformed into bait strains,and reassessed for NO-dependent growth.

NO-Dependent Cell Growth of the ASM/Caspase-3 Clone

Cells were grown in His-Trp-Leu-deficient medium for 72 hrs with orwithout 200 μM DETA-NO(NO). As indicated by an asterisk (FIG. 1B),P<0.001 for ASM/Casp-3 without NO vs. all other comparisons (n=6).

NO-Dependent Interactions Between Caspase-3 and a Set of Novel PartnerProteins (Prey): ASM, IRG, PGM and iNOS

Interactions are revealed as NO-dependent cell growth (FIG. 1C) (singleasterisk, P<0.001 vs. NO-free control, n=6) and as increases inβ-galactosidase activity (FIG. 1D) (single asterisk, P<0.004 vs. NO-freecontrol, n=4). (FIG. 1E) Effect of NO concentration on growth of theASM/caspase-3 clone. Cells were cultured in triple-deficient medium for72 hrs (n=6). (FIG. 1F) Effect of NO concentration on growth of theiNOS/caspase-3 clone (culture conditions as in E) (n=6).

Macrophages exposed to cytokines (tumor necrosis factor andinterleukin-I), a well-established model injury that is apoptotic innature, were used.

Potential redox state-related modifier molecules were determined asbeing nitric oxide, superoxide and hydrogen peroxide, on the basis thatthese were detected as being produced in high levels in macrophagesexposed to cytokines.

It was then established that nitric oxide but not superoxide or hydrogenperoxide is causal to cell injury by demonstration that inhibitors ofnitric oxide protected the cells but inhibitors of superoxide orhydrogen peroxide did not.

It was then determined that nitric oxide, in addition to causing cellinjury, also inhibited proteins involved in protection againstcytokines. This result indicates that non-specific binding of nitricoxide is likely to have some deleterious consequences. Many-foldincreased levels of S-nitrosothiol proteins were measured. Thisindicates that what is needed is a way to identify the S-nitrosylatedproteins that are the targets of nitric oxide and/or the functionalconsequences of these modifications, so these modification can bemanipulated without inhibiting proteins that protect against cytokines.Thus, the goal is to identify the S-nitrosylated proteins that are thetargets of nitric oxide and/or the functional consequences ofmodifications mediated thereby.

For this purpose, protein-protein interactions were determined in ayeast two-hybrid system by the method described in Uetz, P., et al.,Nature 403, 623-627 (2000) and Ito, T., et al., PNAS, 97, 1143-1147(2000). The baits were all known proteins involved in apoptoticcascades. The preys were a library of macrophage genes (mRNA) expressedin response to cytokines that induce apoptosis. Mating pairs ortransfectants were exposed to continuous nitric oxide presence at levelsand flux rates mimicking those which are generated in macrophages, over18 hours (the time course, over which apoptosis occurs in these cells).Room air was used in the determinations to mimic the cell condition atfirst and subsequently low pO₂ (about 5 mm Hg) was used to improvespecificity and increase yield. New partners and/or inhibition ofpartners that were otherwise present, were sought. When caspase was usedas a bait, it was found that nitric oxide induced a novelapoptosis-inducing interaction (based on sequence of the interactingprotein) thus revealing a new and specific approach to inhibitingapoptosis (e.g., drugs that would inhibit interaction between caspaseand the new protein). When a second protein was used, nitric oxideinduced an interaction with a different protein. Use of other baitsresulted in determination of further interactions.

A comparison between a physiological process using the same baits andpreys in a yeast two-hybrid system using the redox state conditions,room air, nanomolar to submicromolar concentration of nitric oxide,nanomolar to micromolar level of nitrosothiols, no reactive oxygenspecies and reducing conditions provided by glutathione and NADH.

In all, 18 new targets were identified in response to nitric oxide.Further, as proof of principle, we have identified one target bylowering the pO₂. In this exemplary case, human red blood cells wereexposed to anaerobiosis or room air and 50 nM NO was added. Low pO₂induced an interaction of a specialized hemoglobin enzyme, thatsubserves NO processing, with an anion exchange protein that wasidentified by co-immunoprecipitation. At low but not high pO₂ thehemoglobin enzyme interacted with the exchanger protein to nitrosylateit. Thus pO₂ can regulate protein-protein interactions. Furthermore,inasmuch as we have established that nitrosylation can promoteprotein-protein interactions, low O₂/NO would operate in concertedfashion to promote multiple protein-protein interactions.

The presence of hydrogen peroxide in the determinations did not produceor modify the majority of determined interactions indicating use of bodyoxygen concentration instead of room air would not have made asignificant difference in those cases and shows specificity fordifferent redox modifiers.

To assess the possibility of NO-dependent regulation of protein-proteininteractions in a cellular context, a modified yeast two-hybridscreening methodology was developed. The first step was to delete theyeast flavohemoglobin gene (Liu et al., 2000), which consumes NO veryefficiently and thus obfuscates NO signaling. The second step was toestablish three complimentary methods to identify NO-dependentprotein-protein interactions, in which NO is delivered from a long-liveddonor, diethyltriamine-nitric oxide (DETA-NO) (half-life ˜18 hours inour assay) dispersed in solid agar (Method 1), soft agar (Method 2) orliquid medium (Method 3), thus covering a range of nitrosylatingconditions (NO flux, gradient and concentration, as well as medium). Itwas also necessary to establish a concentration range of DETA-NO overwhich physiological amounts of NO could be generated in these assayswithout impairing yeast growth (FIG. 1A) (100 μM DETA-NO produces steadystate concentrations of 300 nM NO in yeast culture medium, as determinedusing an NO electrode)

One of the best examples of functional regulation by S-nitrosylation isthe control of caspase-3-dependent death receptor signaling. Inparticular, S-nitrosylation inhibits and denitrosylation facilitates thesequential activation of caspases within macromolecular complexes(Mannick et al., 1999; Dimmeler et al., 1997; Kim et al., 1997).However, the molecular mechanism(s) of action, which enableS-nitrosylation to regulate signal transduction through these complexes,remain poorly understood. Therefore an NO-based two-hybrid screen of acDNA library derived from cytokine-activated murine macrophages (whichare widely used to study the involvement of NO in apoptosis) (Eu et al.,2000) was conducted, using procaspase-3 as bait. PCR analysis withspecific primers verified that the library contained apoptosis-relatedcDNAs including caspase-3,8,9, Apaf-1, Bcl-2 and apoptosis inducingfactor (AIF). Initially 4 million transformants were screened forNO-dependent growth on medium lacking tryptophan, leucine and histidine(9). Thirty-five clones were isolated, which grew in the presence butnot absence of NO, and from which prey plasmids retransfected into thebait strain (clean clones) showed at least 3-fold increases in growth inthe presence of NO, whereas no growth was seen in yeast transformed withbait or prey vector alone. Of the 17 clones showing NO-dependent growtha second time, two clones also showed at least 3-fold activation oflac-Z transcription in the presence vs. absence of NO (p<0.05). Thus,these clones both activated lac-Z transcription and conferred histidineprototrophy in an NO-dependent manner. One of these clones contained apartial sequence (amino acids: 158-927) of the apoptosis-related enzyme,acid sphingomyelinase (ASM) (FIG. 1B). Thus these data establish theprinciple of NO-inducible protein-protein interactions and alsoemphasize the importance of varying nitrosylating conditions in anysystematic analysis of NO-dependent protein-protein interactions.

In order to score the relative dependence on NO of the interactions withcaspase-3, a set of clean clones was generated and assessed forNO-dependent growth (FIG. 1C) and β-gal activity (FIG. 1D). A morerobust overall effect of NO on growth vs. β-gal activity was attributedto the low expression of lac Z in the CG-1945 strain, although it shouldbe noted that the NO-dependent increases in both measures ofprotein-protein interaction were highly reproducible and significant(p<0.001). Growth was particularly robust in the iNOS transformant(˜9-fold), which suggests that the NO-dependent protein-proteininteractions of NOS are of comparatively high-affinity, consistent withan important and previously unsuspected autoregulatory role for NOproduction. Such a role would be analogous to the case of proteinkinases, which have a critical self-regulatory function expressedthrough phosphorylation (Hunter, 2000), and implies that interactionsregulated by NOS in signaling modules may be similarly regulated.

Example 2 Large Scale Yeast Two Hybrid Screening

To enable rapid large-scale screening, an alternative method of genomictwo-hybrid analysis in which E. coli were transformed with plasmidspooled from yeast transformants that were previously grown for severaldays in histidine (HIS)-deficient medium, supplemented continuously withNO (Method 3) was developed. The prey plasmids were isolated andretransfected into caspase-3 bait strains, whose NO-dependent growth wasthen assessed individually. Approximately 1800 E. coli colonies werederived from a screen of ˜4 million transformants; 499 of thesecontained prey plasmids, of which 50 appeared at least twice (asdetermined by HaeIII digestion). Of these, 41 showed NO-dependent growthupon retransformation, and 25 also demonstrated NO-activation of β-galactivity (>3 fold). These clones coded for the immune response gene(IRG), phosphoglycerate mutase (PGM-1), and notably, the induciblenitric oxide synthase (iNOS); the remaining inserts encoded a number ofunknown amino acid sequences that typically terminated with a stopcodon.

As a further means to verify the specificity of the dependence of theinteractions on NO (viz. other redox-related molecules), the same 4clones were examined for the possibility of an interaction dependent onhydrogen peroxide, which was either added repeatedly or generatedcontinuously with glucose oxidase; however, none was found.

Thus, NO is both necessary and sufficient for inducing the interactionsbetween caspase-3 and ASM, IRG, PGM, and iNOS.

Example 3 NO-Dependent Protein Interactions

Methods:

Immunoprecipitation:

Thirty million cells were lysed by gentle homogenization in 1 ml IPbuffer (10 mM NaPi, 100 mM NaCl, 1 mM EDTA, pH 7.9, with proteaseinhibitor cocktail). The supernatant obtained by centrifugation at20,000 g for 10 min was used for immunoprecipitation. Caspase-3immunoprecipitates (2.5 μg anti-caspase-3 monoclonal antibody,Transduction Laboratories) were washed, separated on 10% SDS-PAGE, andblotted with ASM antisera (Santa Cruz Biotechnology); 5% of the samplewas blotted for caspase-3.

ASM activity was measured essentially as described (E. Romiti et al.,2000), using BODIPY FL C₅-sphingomyelin substrate (1.5 nmol) in assaybuffer: 250 mM sodium acetate, pH 5.0, 10 mM EDTA. To assayZn-stimulated ASM activity, 0.1 mM ZnCl was used instead of 10 mM EDTA.30 μl sample was incubated with 70 μl assay buffer at 37° C. for 1-3hours. Reactions were terminated by adding 1.0 ml heptane and 0.29 mlisopropyl alcohol. Phases were separated by adding 0.23 ml H₂O. Theheptane phase washed with 0.23 ml H₂O. The fluorescence of the organicphase (containing BODIPY FL-ceramide) was assayed at 505/514 nmexcitation/emission wavelengths (slit width=2.5).

DETA-NO pretreatment at neutral pH was followed by brief acidification(pH 5.0) to decompose the NO donor. ASM/caspase-3 co-incubations werethen performed at pH 7.2.

ASM Activity is Independent of NO

Purified ASM protein (3.4 μg) was treated with DETA-NO (50 μM-200 μM for30 or 60 min) and ASM activity was measured at pH 5.0 (n=3).

Exposure of caspase-3 to NO induces binding of ASM (n=8, singleasterisk, P<0.002 vs. caspase-3/ASM without NO) (FIG. 2A). Purifiedcaspase-3 was incubated in the presence or absence 20 μM DETA-NO for 60min (30 nM steady state NO), followed by incubation with ASM at 37° C.for 2 hrs, and IP with caspase-3 antibody. Under the same conditions,preincubation of ASM with NO did not induce an interaction withcaspase-3, and the results of incubation of both ASM and caspase-3 withNO were indistinguishable from pre-incubation of caspase-3 alone with NO(not shown). S-nitrosylation of caspase-3 by NO was separately verifiedby photolysis-chemiluminesence (Mannick et al., 1999). The interactionof caspase-3 with ASM was measured as ASM activity in caspase-3 IP's (pH5.0). Little or no ASM activity was present in IP's with caspase-3antibody of either caspase-3 or ASM in isolation.

Amounts of ASM isoforms (47, 57, 75 kDa) in caspase-3 immunoprecipitates(IP) from wild type (WT) vs. nNOS-expressing HEK cells were normalizedwith respect to the amounts of caspase-3 in the IPs (FIG. 2, bargraphs); IPs were performed in the presence and absence of the NOSinhibitor, LNMMA (3 mM). nNOS vs. WT (single asterisk, P<0.01) and nNOSvs. nNOS+LNMMA (double asterisk, p<0.05). A and B, n=7-14; C, n=3-5.

If NO-dependent protein interactions are physiologically relevant, thenthey should be demonstrable in mammalian cells at physiologicallyrelevant NO concentrations. Therefore, the interaction between ASM andiNOS was focused on because of their known functional interrelatednesswith caspase-3 and their role in apoptosis (Mannick et al., 1999; Kim etal., 1997; Eu et al., 2000; Mannick et al., 1994; Bulotta et al., 2001;De Nadai et al., 2000). Human embryonic kidney (HEK-293) cells wereexposed to 200 μM DETA-NO, a concentration that optimally induced inyeast the interactions of caspase-3 with ASM (FIG. 1E) and with iNOS(FIG. 1F). Under these conditions, immunoprecipitation of caspase-3brought down the 57 kD active form of ASM, whereas little or noco-precipitating ASM was seen in the absence of NO (n=4; p<0.03, datanot shown). To define further the NO requirement for this interaction,caspase-3 was immunoprecipitated from wild-type HEK cells and from HEKcells stably transfected with nNOS (Bredt et al., 1992), in the presenceand absence of the NOS inhibitor L-NMMA. Little or no ASM was pulleddown in precipitates from wild-type HEK cells, whereas ASM was readilydetected in the precipitates from nNOS-expressing cells (FIG. 2).Moreover, ASM/caspase-3 interactions were observed not only between the57 kD mature form of ASM, but also with the ASM proform (75 kD) and asmaller processed form (47 kD), which may be alternatively regulated(Ferlinz et al., 1994; Hurwitz et al., 1994). Finally, exposure toL-NMMA significantly reduced the interaction between all isoforms ofacid sphingomyelinase and caspase-3 (p<0.05) (FIG. 2).

Thus, these results demonstrate that the interaction between caspase-3and several differentially processed isoforms of ASM is regulated inmammalian cells by endogenously produced NO. These results furtherindicate that this NO-regulated interaction is reversible.

Covalent modification of proteins by NO may unmask (or alter) proteininteraction domains, and thus alter the affinity of interaction, andconsensus sequences for S-nitrosylation (Stamler et al., 1997) in bothprocaspase-3 and ASM that could underlie the NO-dependence of theirinteraction were identified. The sequences include XCY in primarysequence (but also revealed in tertiary and quaternary structures),where X or Y are acidic and/or basic amino acids (e.g. KRHDE). Anaromatic residue is frequently found in close proximity (in primary ortertiary structure e.g. HCY, in which case an additional acid or basewill typically be present in 3D structure. In addition, cysteinesresiding within hydrophobic pockets of proteins operationally define amotif for S-nitrosylation.

To determine the relevant target(s) of NO, ASM/caspase-3co-precipitations were carried out following NO treatment of eachpartner alone. Using an ASM activity assay, the amounts of ASM thatcould be precipitated with a procaspase-3 antibody was assessed fromco-incubates (2 hours at 37° C.) of ASM (20 μmol) and caspase-3 inlimiting amounts (2.5 μmol). NO pretreatment was for one hour at 25° C.with 20 μM DETA-NO, generating a maximum NO concentration of 30 nM, andwas followed by removal of the NO donor. As shown in FIG. 3A,procaspase-3 that had been pretreated with NO brought down significantlymore ASM than did native procaspase-3 (p<0.002). In contrast, ASM thathad been pretreated with NO did not co-precipitate caspase in increasedamounts (not shown). Control studies showed that NO had no direct effecton ASM activity at these or higher concentrations (FIG. 3B), and thatASM could not be precipitated directly by caspase-3 antibody (FIG. 3A).Thus, it can be concluded that caspase-3 is the relevant target of NOand that modification by NO regulates its interaction with ASM.

Example 4 S-Nitrosylation of Caspase-3

Methods:

Immunoprecipitation:

HEK cell lysates from wild type (WT), nNOS- and eNOS-expressing cellswere immunoprecipitated with caspase-3 monoclonal antibodies and blotted(IB) for NNOS, eNOS, and caspase-3, respectively. eNOS, NNOS andcaspase-3 in whole cell lysates (Lysate) and in IPs from those lysates(7-20% SDS-PAGE) are shown. Data are representative of 5 similarexperiments. eNOS and nNOS were not detectable in control IPs (notshown).

Amounts of eNOS in caspase-3 IPs are dependent on eNOS activity

eNOS-expressing HEK cells were treated with L-arginine (1 mM)/Ca²⁺ (200μM) (to activate NOS) in the presence or absence of L-NMMA (3 mM) (toinhibit NOS activation). Amounts of eNOS were quantified from scannedWestern blots (7-20% SDS-PAGE) and normalized with respect to amounts ofcaspase in the IP (bar graph), (n=3).

It has been shown previously that procaspases in vivo are S-nitrosylatedon their catalytic cysteine and thereby maintained in an inactive state(Mannick et al., 1999; Dimmeler et al., 1997; Kim et al., 1997), butfrom analysis of the crystal structure of caspase-3 (PDB:1PAU), thisactive-site modification would not be anticipated to affectprotein-protein interactions. It is known, however, that caspase-3 canbe S-nitrosylated on more than one cysteine residue (Zech et al., 1999),and in previous experiments, it was observed that a procaspase-3 activesite mutant (C→A) was still S-nitrosylated by NO, and that cleanimmunoprecipitates of this catalytic site mutant from stably transfectedMCF-7 cells showed that it was also nitrosylated in vivo (Mannick etal., 1999). Furthermore, wild-type caspase-3 precipitated from varioushuman cell lines cells may be constitutively nitrosylated at more thanone site. Thus S-nitrosylation of procaspase-3 at an allosteric siteoccurs both in vitro and in vivo.

Taken together with these findings, these results indicate that: 1)procaspase-3 and ASM exhibit a weak binding interaction, and theaffinity of this protein-protein interaction is increased significantlyby low nanomolar amounts of NO both in vitro and in heterologous cellsystems (e.g. yeast); 2) the interaction is regulated reversibly by NOSactivity in mammalian cells; and 3) the mechanism is likely to involveS-nitrosylation of an allosteric site on procaspase-3. This data is thefirst indication that S-nitrosylation of different sites on a proteinmay differentially regulate its function (e.g., allosteric or activesite-thiol modification influences respectively protein-proteininteractions and enzymatic activity).

Inhibition of caspase-3 by S-nitrosylation in multiple cell types and byall three major isoforms of NOS (Mannick et al., 1999; Dimmeler et al.,1997; Kim et al., 1997) suggests that NOSs may share a common motif forrecognition by caspase-3. Notably, the two iNOS clones showing aninteraction with caspase-3 encode a region overlapping the oxygenase,FMN-binding and CaM-binding domains (amino acids 455-647), which isconserved among the three isoforms of NOS. Indeed, as shown in FIG. 4A,caspase-3 antibodies effectively co-precipitated nNOS or eNOS fromHEK-293 cells expressing nNOS or eNOS (Bredt et al., 1992; Sessa et al.,1995). In contrast, isotype-matched control IgG did not precipitatedetectable eNOS or nNOS (not shown). Moreover, it has recently beenshown that regulation of caspase-3 by NOS occurs principally withinmitochondria (Mannick et al., 2001). And in support of a mitochondrialconnection, NOS is enriched in highly purified mitochondrialpreparations from HEK cells and that NOS-dependent protein-proteininteractions are indeed differentially regulated in the mitochondrialand cytosolic fractions.

Example 5 NO-Dependent Protein-Protein Interactions of NOS

Methods:

Yeast Two-Hybrid Assay

Yeast (Δyhb1) were transformed with Gal4 BD-AIF (Bait) and Gal4AD-MIP-1α (Prey). (A) Robust growth of the AIF/MIP-1α clone requires NO(single asterisk, P<0.001 versus AIF/MIP-1α without NO) (n=6). Incontrast, the clone expressing AIF alone shows little growth in eitherthe presence or absence of NO. Cells were grown in His-Trp-Leu-deficientmedium at 30° C. for 72 hrs with or without 200 μM DETA-NO. (B)β-galactosidase activities in samples shown in A (single asterisk,P<0.001 vs. without NO, n=4).

To probe further the physiological relevance of NO-dependentprotein-protein interactions of NOS, the effects of NOS activity onbinding of caspase-3 were assessed. It was found that L-arginine andcalcium supplementation, which activate eNOS, increased its interactionwith caspase-3, while L-NMMA, which inhibits NOS, almost completelyreversed this effect (FIG. 4B). Thus, NOS reversibly regulates its ownbinding to capase-3 through NO production. Although the ramifications ofthese data remain to be explored fully in physiological context,NO-dependent interactions between NOSs and caspase, perhaps involving aternary complex with ASM, would be consistent with the abundantliterature on the regulatory interactions between NOSs, caspases andASM/ceramide in various subcellular compartments (Bulotta et al., 2001;De Nadai et al., 2000; Huwiler et al., 1999; Liu et al., 1995; Igarashiet al., 1999; Takeda et al., 1999; Schwandner et al., 1998).

The demonstration that multiple protein-protein interactions ofcaspase-3 may be determined by NO would have far reaching implicationsfor our understanding of the proteome if extended to other classes ofprotein. To extend the generality of our findings, 2.5 milliontransformants were screened from our macrophage cDNA library forNO-dependent interactions, using apoptosis-inducing factor (AIF) asbait. AIF is implicated in cell death that is independent of caspase-3,and is not known to be regulated by NO (Lipton et al., 2002). Eighteenclones were isolated, of which two showed NO-dependent growth uponretransfection of the prey plasmid into the bait strain (e.g., in twoindependent screens) as well as >3-fold increases in β-gal activity inthe presence of NO. One of these clones contained the entire cDNAsequence of the chemokine, macrophage inflammatory protein-1 alpha(MIP-1α). Since this clone contained a few extra amino acids derivedfrom a linker sequence, the full length MIP-1α coding sequence wasamplified by PCR and cloned into the pGAD10 vector, whose NO-dependentinteraction with AIF was verified. The new clone showed a robust(8-fold) increase in growth in the presence of NO, whereas clonescontaining the bait or prey alone showed virtually no growth either inthe presence of absence of NO (FIG. 5). These data suggest strongly thatNO will be found to regulate a broad spectrum of protein-proteininteractions.

Overall, these findings help to explain a number of experimental andclinical observations: 1) a complex involving NOS/caspase-3 wouldexplain the observation that amounts of NO required to regulatecaspase-3 in vivo are in fact below detection (Mannick et al., 1994); 2)the possibility that caspase-3 might recruit NOS/ASM would rationalizethe observation that NO can co-temporally affect ASM/ceramide andcaspase-3 activities (De Nadai et al., 2000); 3) the novel link betweenAIF and NO could explain NO-mediated apoptosis that is independent ofcaspase-3 (Marshall et al., 2002), and the paradox of pro- andanti-apoptotic effects of AIF reported recently (Lipton et al., 2002);4) the caspase-3/PGM interaction might provide a basis forapoptosis-regulating effects of NO that depend on glycolysis (Almeida etal., 2001); 5) the caspase3/IRG interaction might provide a mechanismfor IRG's role in neural patterning and host defense (Lee et al., 1995;Schmidt and Richter, 2000). In addition, our findings may havetherapeutic implications. For example, production of MIP-1α confersprotection against macrophage trophic HIV (Cocchi et al., 2000), whereasproduction of NO can have the opposite effect (Mannick et al., 1999;Hermann et al., 1997). One reason might be that MIP is sequestered byAIF in NO-generating cells. MIP has also been linked to deregulatedapoptosis in patients with a predisposition to malignancy andprogressive bone marrow aplasia (Haneline et al., 1998), and theNO-dependent interaction with AIF could provide an explanation. Thusthese data might support the use of NOS inhibitors in certain patientswith malignancy, bone marrow failure or infection.

Example 6 Effect of ROSs on Atherosclerosis

Bovine endothelial cells are exposed to LDL cholesterol to induceatherosclerotic changes including production of reactive oxygen species.The predominant reactive oxygen species are identified as beingsuperoxide and hydrogen peroxide, and used in a yeast-2 hybriddetermination at pO₂ of about 70 to identify novel interacting partners,of which one is shown to cause cell death. Inhibiting that new proteinis shown to be anti-atherogenic.

Example 7 Effect of D-Glucose on Endothelial Cell Function

Human umbilical vein endothelial cells are exposed to 30 millimolarD-glucose which impairs endothelial cell function as measured by loss ofnitric oxide bioactivity. Increased oxidative stress is localized to themitochondria using a fluorescent dye and superoxide is shown to be theoxidant (causing the oxidative stress). A yeast two-hybrid determinationusing multiple mitochondrial proteins identifies novel interactions inthe presence of superoxide generated by depleting cells of arginineand/or adding paraquat as compared to determination without superoxidebeing present. In addition, new surface epitopes are found to be presentin cells as identified by antibodies and as described in methodsdescribed above.

Example 8 Effect of Tumor Necrosis Factor (TNF) and Interferon Gamma(IFN-γ) on Protein Expression in Macrophages

Macrophages exposed to TNF and interferon gamma were kept at pO₂ of 5 mmHg. Protein expression was compared in 2D-gels (by differentialprofiling) to pO₂ of 100 mm Hg. A yeast 2 hybrid was then establishedfor the proteins only expressed at low pO₂, showing previouslyunappreciated interactions.

Example 9 Redox Reactions in Pulmonary Fibrosis

The following description is an application of the methods of theinvention for determining protein-protein interactions for apathophysiological process. The pathophysiological process chosen forillustration is interstitial pulmonary fibrosis. Putative RSMMs areidentified based on redox-related enzymes responsible for the generationof reactive oxygen and/or reactive nitrogen species in pulmonaryfibroblast cells, e.g., in response to platelet derived growth factorcausing fibrosis. From these enzymes, the redox-state modifier moleculesthat cause injury and/or the type of protein modification that is mostprevalent are determined. Any protein known to be involved infibroproliferative disorders in the lung is used as bait. All proteinsor genes expressed in pulmonary fibroblast cells (cDNA library) are usedas prey. As a further measure of specificity, only those genes orproteins that are highly expressed in fibroproliferative disorders, asidentified by differential profiling, are utilized. A yeast two-hybridsystem determination is carried out at a body oxygen concentration asdetermined in pulmonary fibrosis tissue with exposing of the system tothe RSMMs identified above as causing injury and/or the type of proteinmodification that is most prevalent, to identify new binding partners,and/or inhibition of other binding, and thus new drug targets.Alternatively, antibodies are generated to cell epitopes before andafter production of reactive oxygen species and new epitopes are therebydiscovered.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. A method for identifying an RSMM inhibited protein complexcomprising: (a) culturing a first cell in a media comprising an RSMM;(b) culturing a second cell in a media without the RSMM; (c) identifyinga first protein complex that exists in the second cell; (d) analyzingthe first cell to determine the existence of a protein complex analogousto the first protein complex; wherein the RSMM inhibited protein complexis identified when the analogous protein complex does not exist orexists to a lesser degree than the first protein complex.
 2. A methodfor identifying an RSMM induced protein complex comprising: (a)culturing a first cell in a media comprising an RSMM; (b) culturing asecond cell in a media without the RSMM; (c) identifying a first proteincomplex that exists in the first cell; (d) analyzing the second cell todetermine the existence of a protein complex analogous to the firstprotein complex; wherein the RSMM induced protein complex is identifiedwhen the analogous protein complex does not exist or exists to a lesserdegree than the first protein complex. 3-4. (canceled)
 5. The method ofclaim 1 in which the RSMM is selected from the group consisting ofnitric oxide, nitric dioxide, dinitrogen trixide, dinitrogen tetraoxide,S-nitrosothiol, nitroxyl anion, HNO, nitrite, nitrate, C—, N, O, S ormetal-nitroso or nitro compounds, hydrogen peroxide, peroxynitrite,other peroxides, alkoxides, superoxide, hypochlorite ion, hydroxylradical and physiological pO₂.
 6. The method of claim 1 in which theRSMM is an NO adduct.
 7. The method of claim 6 wherein the NO adduct isselected from the group consisting of DETA-NO, S-nitrosothiol, SIN-1,angeli's salt, S-nitroso amino acids, S-nitroso-polypeptides, andnitrosoamines.
 8. The method of claim 1 wherein the pO₂ is in a rangefrom about 5 to about 100 mm Hg.
 9. The method of claim 8 wherein therange is from about 10 to about 50 mm Hg.
 10. The method of claim 9wherein the range is from about 10 to about 30 mm Hg. 11-72. (canceled)73. A method for culturing a cell in vitro comprising: (a) providing amedia comprising at least one RSMM; and (b) culturing said cell in themedia.
 74. The method of claim 73 wherein the RSMM is selected from thegroup consisting of nitric oxide, nitric dioxide, hydrogen peroxide,superoxide, hypochlorite ion, hydroxyl radical and physiological pO₂.75. The method of claim 73 wherein the RSMM is an NO adduct.
 76. Themethod of claim 75 wherein the NO adduct is DETA-NO, S-nitrosothiol,S-nitroso amino acids, S-nitroso-polypeptides, and nitrosoamines. 77.The method of claim 74 wherein the pO₂ is in a range from about 5 toabout 100 mm Hg.
 78. The method of claim 77 wherein the range is fromabout 10 to about 30 mm Hg.
 79. The method of claim 78 wherein the rangeis from about 10 to about 30 mm Hg.
 80. The method of claim 73 whereinthe cell is a yeast cell.
 81. The method of claim 80 wherein the yeastcell is S. cerevisiae.
 82. The method of claim 80 wherein the yeast celldoes not express a functional flavohemoglobin gene.
 83. The method ofclaim 73 wherein the concentration of the RSMM is between 100 nM to 1 μMand the time is a period of 24 hours.
 84. The method of claim 73 whereinthe media is a liquid media.
 85. The method of claim 73 wherein themedia is a semisolid media.
 86. The method of claim 73 wherein the mediacomprises between 0.3% to 10% of a solidifying agent.
 87. The method ofclaim 86 wherein the solidifying agent is agar or agarose. 88-163.(canceled)
 164. A method of detecting differences between aprotein-protein interaction within a first cell and a second cell: (a)culturing the first cell in a media comprising an RSMM; (b) isolating aprotein complex from the first cell; and (c) comparing the proteincomplex to a protein complex from the second cell grown in media withoutan RSMM.
 165. The method of claim 164 wherein the first cell and secondcell are mammalian cells.
 166. The method of claim 164 wherein theisolating of the protein complex is by immunoprecipitation.
 167. Themethod of claim 164 wherein the protein complex comprises multiplemembers and at least one member is labeled with a detectable label. 168.The method of claim 167 wherein the detectable label is selected fromthe group consisting of biotin, chemiluminescence, digoxigenin,fluorescence, iodination, kinase, ubiquitin and oligosaccharide. 169.The method of claim 164 wherein the RSMM is selected from the groupconsisting of nitric oxide, nitric dioxide, hydrogen peroxide,superoxide, hypochlorite ion, hydroxyl radical and physiological pO₂.170. The method of claim 164 wherein the RSMM is an NO adduct.
 171. Themethod of claim 170 wherein the NO adduct is DETA-NO, S-nitrosothiol,S-nitroso amino acids, S-nitroso-polypeptides, and nitrosamines. 172.The method of claim 164 wherein the RSMM is pO₂ in a range from about 0to about 100 mm Hg.
 173. The method of claim 172 wherein the range isfrom about 10 to about 50 mm Hg.
 174. The method of claim 173 whereinthe range is about 10 to about 30 mm Hg.
 175. The method of claim 173wherein the range is about 0 to about 20 mm Hg.
 176. The method of claim164 wherein the RSMM is produced by stimulation of an enzyme.
 177. Themethod of claim 176 wherein the stimulation is provided by addition ofcalcium/L-arginine, bradykinin, EGF or other cytokine, growth factor,neuroheumal, or developmental stimulus or activator of a G proteincoupled receptor.
 178. The method of claim 164 wherein the RSMM isproduced from an RSMM-generating enzyme.
 179. The method of claim 178wherein the RSMM-generating enzyme is produced from a recombinantRSMM-generating enzyme vector.
 180. The method of claim 178 wherein theRSMM-generating enzyme is NO synthase, NADPH oxidase, or aconstitutively active rac G-protein. 181-184. (canceled)
 185. The methodof claim 2 in which the RSMM is selected from the group consisting ofnitric oxide, nitric dioxide, dinitrogen trixide, dinitrogen tetraoxide,S-nitrosothiol, nitroxyl anion, HNO, nitrite, nitrate, C—, N, O, S ormetal-nitroso or nitro compounds, hydrogen peroxide, peroxynitrite,other peroxides, alkoxides, superoxide, hypochlorite ion, hydroxylradical and physiological pO₂.
 186. The method of claim 2 in which theRSMM is an NO adduct.
 187. The method of claim 186 wherein the NO adductis selected from the group consisting of DETA-NO, S-nitrosothiol, SIN-1,angeli's salt, S-nitroso amino acids, S-nitroso-polypeptides, andnitrosoamines.
 188. The method of claim 2 wherein the pO₂ is in a rangefrom about 5 to about 100 mm Hg.
 189. The method of claim 188 whereinthe range is from about 10 to about 50 mm Hg.
 190. The method of claim189 wherein the range is from about 10 to about 30 mm Hg.